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Tomato paste supplementation improves endothelial dynamics and reduces plasma total oxidative status in healthy subjects
N U TR IT ION RE S EA RCH 3 2 ( 2 0 12 ) 39 0 –3 94
Available online at www.sciencedirect.com
w w w. n r j o u r n a l . c o m
Tomato paste supplementation improves endothelial dynamics and reduces plasma total oxidative status in healthy subjects Panagiotis Xaplanteris, Charalambos Vlachopoulos⁎, Panagiota Pietri, Dimitrios Terentes-Printzios, Despina Kardara, Nikolaos Alexopoulos, Konstantinos Aznaouridis, Antigoni Miliou, Christodoulos Stefanadis Peripheral Vessels Unit, 1st Cardiology Department, Athens Medical School, Hippokration Hospital, 114, Vasilisis Sofias Ave, 115 27, Athens, Greece
A R T I C LE I N FO
AB S T R A C T
Article history:
Consumption of tomato products is linked to beneficial outcomes through antioxidant and
Received 30 September 2011
anti-inflammatory mechanisms. The aim of this study was to determine whether a 14-day
Revised 18 March 2012
period of tomato paste supplementation would improve endothelial function. Nineteen
Accepted 21 March 2012
volunteers (mean age, 39 ± 13 years; 8 men/11 women) were studied in a randomized (exposure sequence), single-blind (operator), crossover design. The study consisted of a supplementation arm (70 g tomato paste containing 33.3 mg of lycopene) and a control arm,
Keywords:
during which no tomato paste was added to their regular diet. Volunteers maintained their
Endothelial function
regular diet during study arms. Two-week washout periods preceded each arm. Flow-
Lycopene
mediated dilatation (FMD) measured by brachial artery ultrasonography was used as an
Carotenoids
estimate of endothelial function at day 1 (acute response) and day 15 (midterm response).
Tomato
Plasma lipid peroxides were measured with a photometric enzyme-linked immunosorbent
Antioxidant
assay as an index of total oxidative status. Tomato supplementation led to an overall FMD
Crossover studies
increase compared with the control period (P = .047 for repeated-measures 3 × 2 analysis of variance). At day 1, FMD was not significantly increased (P = .329). By day 15, tomato supplementation resulted in an increase in FMD by 3.3% ± 1.4%, whereas at the control arm, FMD declined by −0.5% ± 0.6% (P = .03); magnitudes of change are absolute FMD values. Total oxidative status decreased at the end of the supplementation period compared with baseline values (P = .038). Daily tomato paste consumption exerts a beneficial midterm but not short-term effect on endothelial function. Further studies are warranted to explore the effects of tomato paste on endothelial dilation in different age groups and comorbidities. © 2012 Elsevier Inc. All rights reserved.
1.
Introduction
Several lines of evidence highlight the role of lycopene as an antioxidant. Abundant in tomatoes, lycopene is the most
potent singlet oxygen quencher among carotenoids [1]. Moreover, a number of in vitro studies point to additional anti-inflammatory properties, thus spurring research into its potential role in primary prevention [2]. Nevertheless,
Abbreviations: ANOVA, analysis of variance; CVD, cardiovascular disease; FMD, flow-mediated dilatation; HDL, high-density lipoprotein; LDL, low-density lipoprotein; NMD, nitroglycerin-mediated dilatation; TOS, total oxidative status. ⁎ Corresponding author. Profiti Elia 24, 14575 Athens, Greece. Tel.: +30 6972 272727; fax: +30 210 7473374. E-mail address: [email protected] (C. Vlachopoulos). 0271-5317/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2012.03.011
N U TR IT ION RE S E ARCH 3 2 ( 2 0 12 ) 39 0 –3 9 4
although lycopene plays a clearly beneficial role in the prevention of neoplasias, epidemiologic studies report equivocal results regarding its association with cardiovascular disease (CVD) risk [3–5]. The discrepancy between bench and bedside results can be addressed by assessing early, subclinical manifestations of atherosclerosis. Carotid intima-media thickness, a structural arterial marker, has been inversely linked to lycopene concentrations [6,7]. Trials of short-term supplementation have assessed the impact of lycopene consumption on functional arterial markers such as endothelial function [8] and circulating biomarkers [9], with inconsistent results. The hypothesis of the present study was that a short period of diet supplementation with a tomato-based product would exert a beneficial effect on endothelial function. Nineteen volunteers were studied in a randomized, single-blind, crossover design. Volunteers received a 2-week-long period of tomato paste supplementation and a control procedure with intercalated washout periods. Flow-mediated dilatation (FMD) was used as a marker of endothelial function at baseline and at days 1 and 15 of the supplementation and control arms of the study.
2.
Methods and materials
2.1.
Study population and design
The study population consisted of 19 young, healthy volunteers (age, 39 ± 13 years; 8 men). All were free from CVD, hypertension, diabetes mellitus, dyslipidemia, or family history of premature vascular disease. Seven participants were smokers. Women were examined during the follicular phase of the menstrual cycle; none used oral contraceptives. The study protocol was approved by the Research Ethics Committee of Hippokration Hospital, Athens Medical School, and all subjects gave written informed consent. The study was conducted in a randomized (sequence of exposure), single-blind (operator), crossover design (each subject received both the intervention and control treatments). It consisted of 2 study arms, the tomato paste supplementation arm and the control arm; during the control arm, the volunteers consumed no supplement. Participants were asked to maintain their regular daily dietary pattern for the duration of each study arm; tomato paste was provided in addition to their regular diet during the supplementation arm. Each arm was preceded by a 2-week washout period, during which participants abstained from all lycopene-containing products. Compliance to dietary instructions was evaluated at the end of the study; when participants were asked to report the number of servings of tomato paste they had consumed during the supplementation arm and to return empty cans. Subjects were studied on 3 different occasions for each arm. During the supplementation arm, they were evaluated at baseline, 24 hours after the ingestion of a single dose of tomato paste (short-term response), and at day 15, after the daily ingestion of a dose of tomato paste for 2 weeks (midterm response). Time points for measurements were based on previously published data on lycopene pharmacokinetics in humans [10,11]. Volunteers visited our department at the
391
same time points during the control arm. Subjects abstained from smoking and caffeine/alcohol intake for at least 12 hours before each session. A baseline fasting blood sample was drawn for glucose and lipid profile determination; additional blood samples were drawn at days 1 and 15 of the supplementation period for total oxidative status (TOS) determination. Glucose was measured using the hexokinase method; total cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides were measured using enzymatic colorimetric methods (Abbott ARCHITECT System; Abbott Diagnostics, Abbott Park, Ill). Low-density lipoprotein (LDL) cholesterol was calculated using the Friedewald formula. Total oxidative status was determined by measuring total lipid peroxides in plasma with a photometric enzyme-linked immunosorbent assay (PerOx TOS/total oxidative capacity kit; Immundiagnostik AG, Bensheim, Germany). During the supplementation arm, the participants consumed a commercially available tomato paste (70 g) in the morning after an overnight fast. Products from a single batch were used. The lycopene content of this tomato paste (tomato paste double concentrated 28%; Kyknos SA, Nafplion, Greece) has been previously measured by high-performance liquid chromatography by the manufacturer and was found to be 33.3 mg [12]. The supplied tomato paste also contained 1307 IU of vitamin A, 40.25 mg of vitamin C, 784 μg of β-carotene, 10.5 g of sugar, 11.27 g of total carbohydrate, 2.94 g of protein, 2.45 g of fiber, and 253 kJ of energy (data provided by the manufacturer).
2.2. Measurement of endothelial function of the brachial artery Flow-mediated dilatation is used as an estimate of endothelial function and was measured by high-resolution vascular ultrasound (Agilent Sonos 5500, Hewlett-Packard, Andover, Mass) according to guidelines [13]. Briefly, endotheliumdependent FMD was assessed by measuring the changes in the diameter of the brachial artery for 2 minutes after reactive hyperemia for 5 minutes. Flow-mediated dilatation was defined as the maximum percentage change in brachial artery diameter compared with baseline; that is, FMD = [(postocclusion diameter − resting diameter)/resting diameter] × 100. Reactive hyperemia was calculated as the percentage change of brachial artery blood flow [14]. Nitroglycerin-mediated dilatation (NMD), that is, the endothelial-independent vasodilatation after a sublingual application of 400 μg of nitroglycerin spray, was also assessed at the end of each period. Analyses were conducted offline by 2 different investigators blinded to treatment. The repeatability coefficient for FMD in our unit is 2.06%. It has been previously calculated as defined by the British Standard Institution, according to the following formula: repeatability coefficient = 2 ⁎ √(∑di2/N), where N is the sample size and di is the difference between the 2 measurements in a pair.
2.3.
Statistical analyses
Sample size calculations were based on the data from our unit. The SD of FMD for subjects with characteristics similar to those of our study population was 2.4% [15]. We hypothesized that tomato supplementation would result in an absolute
392
N U TR IT ION RE S EA RCH 3 2 ( 2 0 12 ) 39 0 –3 94
Table 1 – Baseline characteristics and blood measurements of subjects for the supplementation and control periods Tomato supplementation period Age (y) Male/Female BMI (kg/m2) Smokers, n (%) Glucose (mg/dL) Total cholesterol (mg/dL) LDL-cholesterol (mg/dL) HDL-cholesterol (mg/dL) Triglycerides (mg/dL)
Control period
nonparametric Spearman ρ correlation coefficients, owing to a nonnormal distribution of HDL-cholesterol.
P
3.
39 ± 13 8/11 24.8 ± 4.4 7 (36.8) 85 ± 9 180 ± 31
85 ± 9 170 ± 31
.993 .467
113 ± 31
101 ± 26
.456
49 (45-60)
56 ± 5
.008
74 (53-123)
61 (49-73)
.229
Data are presented as means ± SD or median (25th-75th value) for nonnormal distributions and refer to day 0 (before treatment) of each treatment arm. P values refer to the independent-samples t test for normal distributions or Mann-Whitney U test for nonnormal distributions. BMI, body mass index.
increase in FMD of at least 1.5%. Thus, 16 subjects studied in a crossover design would provide 80% power at the 5% level of significance. To ensure the validity of our results, we recruited additional subjects; 19 volunteers participated in the study. Statistical analyses were performed with SPSS 13.0 (SPSS Inc, Chicago, Ill). A 2-tailed P value less than .05 was considered significant. Statistical normality was checked using the Kolmogorov-Smirnov test. Normally distributed continuous variables are presented as the means ± SD; nonnormally distributed variables are presented as the median (25th-75th percentile). Categorical variables are reported as frequencies. The paired-samples t test or Mann-Whitney U tests, where appropriate, were used for analyzing baseline group differences. Changes in continuous variables were analyzed using repeated-measures 2-way analysis of variance (ANOVA). An overall 3 × 2 repeated-measures ANOVA model was used (3 periods [baseline, 1st, 15th day] × 2 interventions [tomato supplementation vs control period]). The values on the 1st and 15th days for FMD, NMD, baseline diameter, reactive hyperemia, and TOS at baseline were further compared using the paired-samples t test. The magnitudes of change for FMD on the 1st and 15th days were calculated as follows: ΔFMD = (FMD value at 1st or 15th days − baseline FMD value), and were compared between arms at each time point using the pairedsamples t test. The magnitudes of change for TOS (ΔTOS) were similarly calculated for days 1 and 15 of the supplementation period. Possible carryover effects were tested by the independent-samples t test, with ΔFMD at each time point and each arm as the dependent variable and the sequence of received treatments (tomato supplementation followed by control vs the opposite sequence) as the grouping variable. The correlation of baseline FMD values with ΔFMD at days 1 and 15 of the supplementation period was tested by calculating Pearson r correlation coefficients. The correlation of baseline HDLcholesterol values at the start of the supplementation period with ΔFMD at days 1 and 15 was tested by calculating the
Results
The baseline characteristics of the study participants are presented in Table 1. Baseline glucose and lipid profiles did not differ between periods, except for higher HDL values in the control period. All 19 participants reported that they had consumed all of the provided servings of tomato paste during the supplementation period. The tomato supplementation led to an overall increase in FMD across time points when compared with the control period (P = .047 for the overall time × treatment withinsubjects effect) (Table 2). The effect of supplementation and control periods on FMD is best described by the magnitude of change. At day 1, FMD was increased by 1.4% ± 1.5% at the supplementation arm and decreased by −0.3% ± 0.6% at the control arm; these changes were not significantly different from each other (P = .329). By day 15, tomato supplementation resulted in an increase in FMD by 3.3% ± 1.4%, and at the control arm, FMD values declined by −0.5% ± 0.6%; the difference between the magnitudes of change at day 15 was statistically significant (P = .03). (All the aforementioned magnitudes of change are absolute FMD values.) Due to the crossover design of the study, there is a possibility of carryover effects. Four independent-samples t tests were performed, with sequence of treatments as the grouping variable and ΔFMD at days 1 and 15 for the supplementation and control arm as dependent variables. There was no significant difference in ΔFMD at day 1 for either sequence (P = .383 for the supplementation arm, P = .517 for the control arm). Similar results were obtained for ΔFMD at day 15 (P = .104 for the supplementation arm, P = .141 for the control arm). Baseline FMD values showed a negative correlation with ΔFMD at day 1 (r = −0.826, P = .001) and day 15 (r = −0.832, P = .001), indicating a pronounced beneficial effect of tomato paste consumption at lower baseline FMD values. To examine the effect of baseline HDL-cholesterol values on ΔFMD at days 1 and 15, correlation coefficients were calculated. There was a positive correlation between baseline HDL-cholesterol and ΔFMD at day 1 (ρ = 0.611, P = .016). Nevertheless, baseline HDL-cholesterol and ΔFMD at day 15 did not correlate (ρ = 0.120, P = .67). Nitroglycerin-mediated dilatation, baseline brachial artery diameter, and reactive hyperemia did not change across time points in either group (Table 2). Total oxidative status did not change at day 1 (P = .666) but decreased at the end of the supplementation period compared with baseline values (P = .038). Both ΔTOS and ΔFMD at day 15 of the supplementation period were inversely correlated (ρ = −0.731, P = .001), indicating that improvement of endothelial function correlated with diminishing TOS values.
4.
Discussion
This is the first study to investigate the endothelial effects of tomato paste administration after a short-term and midterm
393
N U TR IT ION RE S E ARCH 3 2 ( 2 0 12 ) 39 0 –3 9 4
Table 2 – Dependent variable values across time points for the supplementation and control periods Tomato supplementation period Baseline FMD (%) NMD (%) Baseline diameter (mm) Reactive hyperemia (%) TOS (μmol/L)
4.2 15.5 2.94 541 362
± ± ± ± ±
5.1 6.1 0.61 267 241
1st day 5.6 16.2 2.95 552 348
± ± ± ± ±
3.9 6.1 0.61 250 248
15th day 7.5 14.6 2.93 569 300
± ± ± ± ±
3.5 ⁎, ⁎⁎ 6.1 0.83 292 253⁎
Control period Baseline 5.0 13.6 2.99 438
± ± ± ±
3.5 10.0 0.57 289
1st day 4.7 15.2 2.93 506
± ± ± ±
3.5 10.5 0.57 320
15th day 4.5 12.6 2.99 559
± ± ± ±
3.5 8.3 0.52 296
P value for repeated-measures ANOVA .047 .358 .592 .210
Data are presented as means ± SD. The last column reports P values for the overall 3 × 2 repeated-measures ANOVA model (3 time points × 2 interventions). Mean FMD and NMD values reported are absolute values at each time point. During the tomato supplementation period, the volunteers consumed a portion of tomato paste (70g) every morning for 2 weeks; no supplementary tomato paste was consumed during the control period. ⁎ P < .05 vs baseline. ⁎⁎ P = .05 vs 1st day for paired-samples t test comparisons between 2 time points.
(1 and 14 days, respectively) period of dietary supplementation in young, healthy volunteers. As hypothesized, tomato paste exerted a beneficial effect on the endothelium as demonstrated by the rise in FMD. However, the effect was observed only after a supplementation period of 2 weeks, not after a single portion. Importantly, the possibility of carryover effects between study arms due to the crossover design of the study has been excluded. These results extend previous findings from our unit regarding the impact of dietary [14,16] and lifestyle [17,18] factors on arterial function [19,20]. Our study may have important clinical implications. Endothelial function is affected favorably with adherence to a Mediterranean diet pattern [21]; this is clinically relevant in the setting of obesity [22]. Taken together, these findings suggest that tomato products may act synergistically with other components of the Mediterranean diet (red wine, olive oil) to promote vascular health. Our findings suggest that tomato paste improves endothelial function independently from baseline HDL-cholesterol values. Moreover, we have observed an inverse relationship between baseline FMD values and ΔFMD. This emphasizes a potential for a pronounced beneficial role for individuals with impaired endothelial function (smokers, patients with dyslipidemia, diabetic patients, hypertensive patients). These findings raise the possibility of using tomato products as nonpharmacologic adjuncts for restoring endothelial dysfunction, an established barometer of CVD risk. Published studies are inconclusive regarding the endothelial potential of tomatoes and lycopene. Stangl et al [8] have reported a lack of effect of tomato puree administration on FMD in healthy, postmenopausal women; nevertheless, the duration of supplementation was short (7 days). In contrast, Kim et al [23] reported that endothelial function and a series of biomarkers improve after an 8-week period of lycopene supplementation. Multiple mechanisms contribute to the effect of tomato products. The antioxidant properties of carotenoids, mainly lycopene, are central to their beneficial properties. The processing of tomatoes and cooking them with olive oil increases the carotenoid bioavailability. Carotenoids prevent the oxidation of LDL-cholesterol, and some studies report a mild LDL-cholesterol reduction effect after tomato juice
supplementation [24]. Moreover, anti-inflammatory benefits have been ascribed to tomato products; however, published data are conflicting [2,25]. Plasma lipid peroxides were measured as a cumulative marker of TOS. The decline in TOS at the end of the supplementation period demonstrates the midterm antioxidant potential of tomato paste. Nevertheless, TOS did not change acutely after ingestion of the first serving. The concurrent TOS reduction and improvement in FMD at day 15 could be explained through a cause-and-effect mechanism. Thus, ingestion of tomato paste abundant in antioxidants could decrease TOS, resulting in a favorable redox state for vessel walls, which would increase the production of vasodilatory molecules (nitric oxide, adenosine). However plausible this scenario is, it cannot be substantiated by the nature of our in vivo study. Confirmation of this mechanism would require ex vivo protocols. We chose to use a commercially available tomato product instead of pharmaceutical preparations of lycopene. This allows for the direct extrapolation of our findings to everyday culinary practices. The amount of lycopene contained in a serving of tomato paste exceeds the average dietary intake; nonetheless, it represents a physiologically relevant dose that compares favorably to doses used in similar human studies in the past [8,10]. The beneficial effect of tomato products may be attributed to an additive/synergistic effect of lycopene with other micronutrients, including α-tocopherol, vitamin C, and rutin [26]. The decision to provide the tomato paste in the morning was based on the pharmacokinetics of lycopene. Tomato products are usually consumed during lunch and/or dinner; heating during cooking increases lycopene bioavailability by releasing it from the food matrix. Concomitant fat consumption may also improve bioavailability because lycopene is lipid soluble. It is plausible that consumption at lunch or dinnertime could have magnified the salutary impact on endothelial function [11]. Some limitations of our study should be acknowledged. Lycopene concentrations were not measured in blood samples because the necessary high-performance liquid chromatography techniques are not available to us. Total oxidative status was not measured for the control arm. A fixed dose of
394
N U TR IT ION RE S EA RCH 3 2 ( 2 0 12 ) 39 0 –3 94
400 μg of nitroglycerin was used during NMD measurements. Confirmatory research is also needed to evaluate the role of the dietary portions of tomatoes; we used an amount of lycopene that exceeds the average daily intake. The inclusion of smokers could have blunted the beneficial effects of tomato paste, given the effect of smoking on the antioxidant properties of bioactive compounds. In conclusion, we have demonstrated that a midterm tomato dietary supplementation period improved endothelial function in a group of healthy young men and women. Individuals with lower baseline FMD values were particularly benefited, as demonstrated by the fact that they exhibited greater increases in FMD values after consumption of tomato paste. Further research is warranted to ascertain the impact of long-term tomato supplementation on endothelial function and CVD risk in various clinical settings.
Acknowledgment The study was funded by Athens Medical School, Greece. There are no conflicts of interest to disclose pertaining to this study.
REFERENCES [1] Miller NJ, Sampson J, Candeias LP, Bramley PM, Rice-Evans CA. Antioxidant activities of carotenes and xanthophylls. FEBS Lett 1996;384:240–2. [2] Hung CF, Huang TF, Chen BH, Shieh JM, Wu PH, Wu WB. Lycopene inhibits TNF-alpha–induced endothelial ICAM-1 expression and monocyte-endothelial adhesion. Eur J Pharmacol 2008;586:275–82. [3] Sesso HD, Buring JE, Norkus EP, Gaziano JM. Plasma lycopene, other carotenoids, and retinol and the risk of cardiovascular disease in men. Am J Clin Nutr 2005;81:990–7. [4] Kohlmeier L, Kark JD, Gomez-Gracia E, Martin BC, Steck SE, Kardinaal AF, et al. Lycopene and myocardial infarction risk in the EURAMIC Study. Am J Epidemiol 1997;146:618–26. [5] Hozawa A, Jacobs Jr DR, Steffes MW, Gross MD, Steffen LM, Lee DH. Relationships of circulating carotenoid concentrations with several markers of inflammation, oxidative stress, and endothelial dysfunction: the Coronary Artery Risk Development in Young Adults (CARDIA)/Young Adult Longitudinal Trends in Antioxidants (YALTA) study. Clin Chem 2007;53:447–55. [6] Rissanen T, Voutilainen S, Nyyssonen K, Salonen R, Salonen JT. Low plasma lycopene concentration is associated with increased intima-media thickness of the carotid artery wall. Arterioscler Thromb Vasc Biol 2000;20:2677–81. [7] Gianetti J, Pedrinelli R, Petrucci R, Lazzerini G, De Caterina M, Bellomo G, et al. Inverse association between carotid intima-media thickness and the antioxidant lycopene in atherosclerosis. Am Heart J 2002;143:467–74. [8] Stangl V, Kuhn C, Hentschel S, Jochmann N, Jacob C, Bohm V, et al. Lack of effects of tomato products on endothelial function in human subjects: results of a randomised, placebocontrolled cross-over study. Br J Nutr 2011;105:263–7. [9] Denniss SG, Haffner TD, Kroetsch JT, Davidson SR, Rush JW, Hughson RL. Effect of short-term lycopene supplementation and postprandial dyslipidemia on plasma antioxidants and biomarkers of endothelial health in young, healthy individuals. Vasc Health Risk Manag 2008;4:213–22.
[10] Diwadkar-Navsariwala V, Novotny JA, Gustin DM, Sosman JA, Rodvold KA, Crowell JA, et al. A physiological pharmacokinetic model describing the disposition of lycopene in healthy men. J Lipid Res 2003;44:1927–39. [11] Frohlich K, Kaufmann K, Bitsch R, Bohm V. Effects of ingestion of tomatoes, tomato juice and tomato puree on contents of lycopene isomers, tocopherols and ascorbic acid in human plasma as well as on lycopene isomer pattern. Br J Nutr 2006;95:734–41. [12] Porrini M, Riso P, Brusamolino A, Berti C, Guarnieri S, Visioli F. Daily intake of a formulated tomato drink affects carotenoid plasma and lymphocyte concentrations and improves cellular antioxidant protection. Br J Nutr 2005;93:93–9. [13] Corretti MC, Anderson TJ, Benjamin EJ, Celermajer D, Charbonneau F, Creager MA, et al. Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force. J Am Coll Cardiol 2002;39:257–65. [14] Alexopoulos N, Vlachopoulos C, Aznaouridis K, Baou K, Vasiliadou C, Pietri P, et al. The acute effect of green tea consumption on endothelial function in healthy individuals. Eur J Cardiovasc Prev Rehabil 2008;15:300–5. [15] Vlachopoulos C, Aznaouridis K, Dagre A, Vasiliadou C, Masoura C, Stefanadi E, et al. Protective effect of atorvastatin on acute systemic inflammation-induced endothelial dysfunction in hypercholesterolaemic subjects. Eur Heart J 2007;28:2102–9. [16] Vlachopoulos C, Aznaouridis K, Alexopoulos N, Economou E, Andreadou I, Stefanadis C. Effect of dark chocolate on arterial function in healthy individuals. Am J Hypertens 2005;18: 785–91. [17] Vlachopoulos C, Xaplanteris P, Stefanadis C. Mental stress, arterial stiffness, central pressures, and cardiovascular risk. Hypertension 2010;56:e28 [author reply e9]. [18] Vlachopoulos C, Xaplanteris P, Alexopoulos N, Aznaouridis K, Vasiliadou C, Baou K, et al. Divergent effects of laughter and mental stress on arterial stiffness and central hemodynamics. Psychosom Med 2009;71:446–53. [19] Vlachopoulos C, Aznaouridis K, Stefanadis C. Prediction of cardiovascular events and all-cause mortality with arterial stiffness: a systematic review and meta-analysis. J Am Coll Cardiol 2010;55:1318–27. [20] Vlachopoulos C, Alexopoulos N, Stefanadis C. Aortic stiffness: prime time for integration into clinical practice? Hellenic J Cardiol 2010;51:385–90. [21] Marin C, Ramirez R, Delgado-Lista J, Yubero-Serrano EM, Perez-Martinez P, Carracedo J, et al. Mediterranean diet reduces endothelial damage and improves the regenerative capacity of endothelium. Am J Clin Nutr 2011;93: 267–74. [22] Rallidis LS, Lekakis J, Kolomvotsou A, Zampelas A, Vamvakou G, Efstathiou S, et al. Close adherence to a Mediterranean diet improves endothelial function in subjects with abdominal obesity. Am J Clin Nutr 2009;90:263–8. [23] Kim JY, Paik JK, Kim OY, Park HW, Lee JH, Jang Y. Effects of lycopene supplementation on oxidative stress and markers of endothelial function in healthy men. Atherosclerosis 2011;215:189–95. [24] Silaste ML, Alfthan G, Aro A, Kesaniemi YA, Horkko S. Tomato juice decreases LDL cholesterol levels and increases LDL resistance to oxidation. Br J Nutr 2007;98:1251–8. [25] Blum A, Monir M, Khazim K, Peleg A, Blum N. Tomato-rich (Mediterranean) diet does not modify inflammatory markers. Clin Invest Med 2007;30:E70–4. [26] Erdman Jr JW, Ford NA, Lindshield BL. Are the health attributes of lycopene related to its antioxidant function? Arch Biochem Biophys 2009;483:229–35.
Available online at www.sciencedirect.com
w w w. n r j o u r n a l . c o m
Tomato paste supplementation improves endothelial dynamics and reduces plasma total oxidative status in healthy subjects Panagiotis Xaplanteris, Charalambos Vlachopoulos⁎, Panagiota Pietri, Dimitrios Terentes-Printzios, Despina Kardara, Nikolaos Alexopoulos, Konstantinos Aznaouridis, Antigoni Miliou, Christodoulos Stefanadis Peripheral Vessels Unit, 1st Cardiology Department, Athens Medical School, Hippokration Hospital, 114, Vasilisis Sofias Ave, 115 27, Athens, Greece
A R T I C LE I N FO
AB S T R A C T
Article history:
Consumption of tomato products is linked to beneficial outcomes through antioxidant and
Received 30 September 2011
anti-inflammatory mechanisms. The aim of this study was to determine whether a 14-day
Revised 18 March 2012
period of tomato paste supplementation would improve endothelial function. Nineteen
Accepted 21 March 2012
volunteers (mean age, 39 ± 13 years; 8 men/11 women) were studied in a randomized (exposure sequence), single-blind (operator), crossover design. The study consisted of a supplementation arm (70 g tomato paste containing 33.3 mg of lycopene) and a control arm,
Keywords:
during which no tomato paste was added to their regular diet. Volunteers maintained their
Endothelial function
regular diet during study arms. Two-week washout periods preceded each arm. Flow-
Lycopene
mediated dilatation (FMD) measured by brachial artery ultrasonography was used as an
Carotenoids
estimate of endothelial function at day 1 (acute response) and day 15 (midterm response).
Tomato
Plasma lipid peroxides were measured with a photometric enzyme-linked immunosorbent
Antioxidant
assay as an index of total oxidative status. Tomato supplementation led to an overall FMD
Crossover studies
increase compared with the control period (P = .047 for repeated-measures 3 × 2 analysis of variance). At day 1, FMD was not significantly increased (P = .329). By day 15, tomato supplementation resulted in an increase in FMD by 3.3% ± 1.4%, whereas at the control arm, FMD declined by −0.5% ± 0.6% (P = .03); magnitudes of change are absolute FMD values. Total oxidative status decreased at the end of the supplementation period compared with baseline values (P = .038). Daily tomato paste consumption exerts a beneficial midterm but not short-term effect on endothelial function. Further studies are warranted to explore the effects of tomato paste on endothelial dilation in different age groups and comorbidities. © 2012 Elsevier Inc. All rights reserved.
1.
Introduction
Several lines of evidence highlight the role of lycopene as an antioxidant. Abundant in tomatoes, lycopene is the most
potent singlet oxygen quencher among carotenoids [1]. Moreover, a number of in vitro studies point to additional anti-inflammatory properties, thus spurring research into its potential role in primary prevention [2]. Nevertheless,
Abbreviations: ANOVA, analysis of variance; CVD, cardiovascular disease; FMD, flow-mediated dilatation; HDL, high-density lipoprotein; LDL, low-density lipoprotein; NMD, nitroglycerin-mediated dilatation; TOS, total oxidative status. ⁎ Corresponding author. Profiti Elia 24, 14575 Athens, Greece. Tel.: +30 6972 272727; fax: +30 210 7473374. E-mail address: [email protected] (C. Vlachopoulos). 0271-5317/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2012.03.011
N U TR IT ION RE S E ARCH 3 2 ( 2 0 12 ) 39 0 –3 9 4
although lycopene plays a clearly beneficial role in the prevention of neoplasias, epidemiologic studies report equivocal results regarding its association with cardiovascular disease (CVD) risk [3–5]. The discrepancy between bench and bedside results can be addressed by assessing early, subclinical manifestations of atherosclerosis. Carotid intima-media thickness, a structural arterial marker, has been inversely linked to lycopene concentrations [6,7]. Trials of short-term supplementation have assessed the impact of lycopene consumption on functional arterial markers such as endothelial function [8] and circulating biomarkers [9], with inconsistent results. The hypothesis of the present study was that a short period of diet supplementation with a tomato-based product would exert a beneficial effect on endothelial function. Nineteen volunteers were studied in a randomized, single-blind, crossover design. Volunteers received a 2-week-long period of tomato paste supplementation and a control procedure with intercalated washout periods. Flow-mediated dilatation (FMD) was used as a marker of endothelial function at baseline and at days 1 and 15 of the supplementation and control arms of the study.
2.
Methods and materials
2.1.
Study population and design
The study population consisted of 19 young, healthy volunteers (age, 39 ± 13 years; 8 men). All were free from CVD, hypertension, diabetes mellitus, dyslipidemia, or family history of premature vascular disease. Seven participants were smokers. Women were examined during the follicular phase of the menstrual cycle; none used oral contraceptives. The study protocol was approved by the Research Ethics Committee of Hippokration Hospital, Athens Medical School, and all subjects gave written informed consent. The study was conducted in a randomized (sequence of exposure), single-blind (operator), crossover design (each subject received both the intervention and control treatments). It consisted of 2 study arms, the tomato paste supplementation arm and the control arm; during the control arm, the volunteers consumed no supplement. Participants were asked to maintain their regular daily dietary pattern for the duration of each study arm; tomato paste was provided in addition to their regular diet during the supplementation arm. Each arm was preceded by a 2-week washout period, during which participants abstained from all lycopene-containing products. Compliance to dietary instructions was evaluated at the end of the study; when participants were asked to report the number of servings of tomato paste they had consumed during the supplementation arm and to return empty cans. Subjects were studied on 3 different occasions for each arm. During the supplementation arm, they were evaluated at baseline, 24 hours after the ingestion of a single dose of tomato paste (short-term response), and at day 15, after the daily ingestion of a dose of tomato paste for 2 weeks (midterm response). Time points for measurements were based on previously published data on lycopene pharmacokinetics in humans [10,11]. Volunteers visited our department at the
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same time points during the control arm. Subjects abstained from smoking and caffeine/alcohol intake for at least 12 hours before each session. A baseline fasting blood sample was drawn for glucose and lipid profile determination; additional blood samples were drawn at days 1 and 15 of the supplementation period for total oxidative status (TOS) determination. Glucose was measured using the hexokinase method; total cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides were measured using enzymatic colorimetric methods (Abbott ARCHITECT System; Abbott Diagnostics, Abbott Park, Ill). Low-density lipoprotein (LDL) cholesterol was calculated using the Friedewald formula. Total oxidative status was determined by measuring total lipid peroxides in plasma with a photometric enzyme-linked immunosorbent assay (PerOx TOS/total oxidative capacity kit; Immundiagnostik AG, Bensheim, Germany). During the supplementation arm, the participants consumed a commercially available tomato paste (70 g) in the morning after an overnight fast. Products from a single batch were used. The lycopene content of this tomato paste (tomato paste double concentrated 28%; Kyknos SA, Nafplion, Greece) has been previously measured by high-performance liquid chromatography by the manufacturer and was found to be 33.3 mg [12]. The supplied tomato paste also contained 1307 IU of vitamin A, 40.25 mg of vitamin C, 784 μg of β-carotene, 10.5 g of sugar, 11.27 g of total carbohydrate, 2.94 g of protein, 2.45 g of fiber, and 253 kJ of energy (data provided by the manufacturer).
2.2. Measurement of endothelial function of the brachial artery Flow-mediated dilatation is used as an estimate of endothelial function and was measured by high-resolution vascular ultrasound (Agilent Sonos 5500, Hewlett-Packard, Andover, Mass) according to guidelines [13]. Briefly, endotheliumdependent FMD was assessed by measuring the changes in the diameter of the brachial artery for 2 minutes after reactive hyperemia for 5 minutes. Flow-mediated dilatation was defined as the maximum percentage change in brachial artery diameter compared with baseline; that is, FMD = [(postocclusion diameter − resting diameter)/resting diameter] × 100. Reactive hyperemia was calculated as the percentage change of brachial artery blood flow [14]. Nitroglycerin-mediated dilatation (NMD), that is, the endothelial-independent vasodilatation after a sublingual application of 400 μg of nitroglycerin spray, was also assessed at the end of each period. Analyses were conducted offline by 2 different investigators blinded to treatment. The repeatability coefficient for FMD in our unit is 2.06%. It has been previously calculated as defined by the British Standard Institution, according to the following formula: repeatability coefficient = 2 ⁎ √(∑di2/N), where N is the sample size and di is the difference between the 2 measurements in a pair.
2.3.
Statistical analyses
Sample size calculations were based on the data from our unit. The SD of FMD for subjects with characteristics similar to those of our study population was 2.4% [15]. We hypothesized that tomato supplementation would result in an absolute
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Table 1 – Baseline characteristics and blood measurements of subjects for the supplementation and control periods Tomato supplementation period Age (y) Male/Female BMI (kg/m2) Smokers, n (%) Glucose (mg/dL) Total cholesterol (mg/dL) LDL-cholesterol (mg/dL) HDL-cholesterol (mg/dL) Triglycerides (mg/dL)
Control period
nonparametric Spearman ρ correlation coefficients, owing to a nonnormal distribution of HDL-cholesterol.
P
3.
39 ± 13 8/11 24.8 ± 4.4 7 (36.8) 85 ± 9 180 ± 31
85 ± 9 170 ± 31
.993 .467
113 ± 31
101 ± 26
.456
49 (45-60)
56 ± 5
.008
74 (53-123)
61 (49-73)
.229
Data are presented as means ± SD or median (25th-75th value) for nonnormal distributions and refer to day 0 (before treatment) of each treatment arm. P values refer to the independent-samples t test for normal distributions or Mann-Whitney U test for nonnormal distributions. BMI, body mass index.
increase in FMD of at least 1.5%. Thus, 16 subjects studied in a crossover design would provide 80% power at the 5% level of significance. To ensure the validity of our results, we recruited additional subjects; 19 volunteers participated in the study. Statistical analyses were performed with SPSS 13.0 (SPSS Inc, Chicago, Ill). A 2-tailed P value less than .05 was considered significant. Statistical normality was checked using the Kolmogorov-Smirnov test. Normally distributed continuous variables are presented as the means ± SD; nonnormally distributed variables are presented as the median (25th-75th percentile). Categorical variables are reported as frequencies. The paired-samples t test or Mann-Whitney U tests, where appropriate, were used for analyzing baseline group differences. Changes in continuous variables were analyzed using repeated-measures 2-way analysis of variance (ANOVA). An overall 3 × 2 repeated-measures ANOVA model was used (3 periods [baseline, 1st, 15th day] × 2 interventions [tomato supplementation vs control period]). The values on the 1st and 15th days for FMD, NMD, baseline diameter, reactive hyperemia, and TOS at baseline were further compared using the paired-samples t test. The magnitudes of change for FMD on the 1st and 15th days were calculated as follows: ΔFMD = (FMD value at 1st or 15th days − baseline FMD value), and were compared between arms at each time point using the pairedsamples t test. The magnitudes of change for TOS (ΔTOS) were similarly calculated for days 1 and 15 of the supplementation period. Possible carryover effects were tested by the independent-samples t test, with ΔFMD at each time point and each arm as the dependent variable and the sequence of received treatments (tomato supplementation followed by control vs the opposite sequence) as the grouping variable. The correlation of baseline FMD values with ΔFMD at days 1 and 15 of the supplementation period was tested by calculating Pearson r correlation coefficients. The correlation of baseline HDLcholesterol values at the start of the supplementation period with ΔFMD at days 1 and 15 was tested by calculating the
Results
The baseline characteristics of the study participants are presented in Table 1. Baseline glucose and lipid profiles did not differ between periods, except for higher HDL values in the control period. All 19 participants reported that they had consumed all of the provided servings of tomato paste during the supplementation period. The tomato supplementation led to an overall increase in FMD across time points when compared with the control period (P = .047 for the overall time × treatment withinsubjects effect) (Table 2). The effect of supplementation and control periods on FMD is best described by the magnitude of change. At day 1, FMD was increased by 1.4% ± 1.5% at the supplementation arm and decreased by −0.3% ± 0.6% at the control arm; these changes were not significantly different from each other (P = .329). By day 15, tomato supplementation resulted in an increase in FMD by 3.3% ± 1.4%, and at the control arm, FMD values declined by −0.5% ± 0.6%; the difference between the magnitudes of change at day 15 was statistically significant (P = .03). (All the aforementioned magnitudes of change are absolute FMD values.) Due to the crossover design of the study, there is a possibility of carryover effects. Four independent-samples t tests were performed, with sequence of treatments as the grouping variable and ΔFMD at days 1 and 15 for the supplementation and control arm as dependent variables. There was no significant difference in ΔFMD at day 1 for either sequence (P = .383 for the supplementation arm, P = .517 for the control arm). Similar results were obtained for ΔFMD at day 15 (P = .104 for the supplementation arm, P = .141 for the control arm). Baseline FMD values showed a negative correlation with ΔFMD at day 1 (r = −0.826, P = .001) and day 15 (r = −0.832, P = .001), indicating a pronounced beneficial effect of tomato paste consumption at lower baseline FMD values. To examine the effect of baseline HDL-cholesterol values on ΔFMD at days 1 and 15, correlation coefficients were calculated. There was a positive correlation between baseline HDL-cholesterol and ΔFMD at day 1 (ρ = 0.611, P = .016). Nevertheless, baseline HDL-cholesterol and ΔFMD at day 15 did not correlate (ρ = 0.120, P = .67). Nitroglycerin-mediated dilatation, baseline brachial artery diameter, and reactive hyperemia did not change across time points in either group (Table 2). Total oxidative status did not change at day 1 (P = .666) but decreased at the end of the supplementation period compared with baseline values (P = .038). Both ΔTOS and ΔFMD at day 15 of the supplementation period were inversely correlated (ρ = −0.731, P = .001), indicating that improvement of endothelial function correlated with diminishing TOS values.
4.
Discussion
This is the first study to investigate the endothelial effects of tomato paste administration after a short-term and midterm
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Table 2 – Dependent variable values across time points for the supplementation and control periods Tomato supplementation period Baseline FMD (%) NMD (%) Baseline diameter (mm) Reactive hyperemia (%) TOS (μmol/L)
4.2 15.5 2.94 541 362
± ± ± ± ±
5.1 6.1 0.61 267 241
1st day 5.6 16.2 2.95 552 348
± ± ± ± ±
3.9 6.1 0.61 250 248
15th day 7.5 14.6 2.93 569 300
± ± ± ± ±
3.5 ⁎, ⁎⁎ 6.1 0.83 292 253⁎
Control period Baseline 5.0 13.6 2.99 438
± ± ± ±
3.5 10.0 0.57 289
1st day 4.7 15.2 2.93 506
± ± ± ±
3.5 10.5 0.57 320
15th day 4.5 12.6 2.99 559
± ± ± ±
3.5 8.3 0.52 296
P value for repeated-measures ANOVA .047 .358 .592 .210
Data are presented as means ± SD. The last column reports P values for the overall 3 × 2 repeated-measures ANOVA model (3 time points × 2 interventions). Mean FMD and NMD values reported are absolute values at each time point. During the tomato supplementation period, the volunteers consumed a portion of tomato paste (70g) every morning for 2 weeks; no supplementary tomato paste was consumed during the control period. ⁎ P < .05 vs baseline. ⁎⁎ P = .05 vs 1st day for paired-samples t test comparisons between 2 time points.
(1 and 14 days, respectively) period of dietary supplementation in young, healthy volunteers. As hypothesized, tomato paste exerted a beneficial effect on the endothelium as demonstrated by the rise in FMD. However, the effect was observed only after a supplementation period of 2 weeks, not after a single portion. Importantly, the possibility of carryover effects between study arms due to the crossover design of the study has been excluded. These results extend previous findings from our unit regarding the impact of dietary [14,16] and lifestyle [17,18] factors on arterial function [19,20]. Our study may have important clinical implications. Endothelial function is affected favorably with adherence to a Mediterranean diet pattern [21]; this is clinically relevant in the setting of obesity [22]. Taken together, these findings suggest that tomato products may act synergistically with other components of the Mediterranean diet (red wine, olive oil) to promote vascular health. Our findings suggest that tomato paste improves endothelial function independently from baseline HDL-cholesterol values. Moreover, we have observed an inverse relationship between baseline FMD values and ΔFMD. This emphasizes a potential for a pronounced beneficial role for individuals with impaired endothelial function (smokers, patients with dyslipidemia, diabetic patients, hypertensive patients). These findings raise the possibility of using tomato products as nonpharmacologic adjuncts for restoring endothelial dysfunction, an established barometer of CVD risk. Published studies are inconclusive regarding the endothelial potential of tomatoes and lycopene. Stangl et al [8] have reported a lack of effect of tomato puree administration on FMD in healthy, postmenopausal women; nevertheless, the duration of supplementation was short (7 days). In contrast, Kim et al [23] reported that endothelial function and a series of biomarkers improve after an 8-week period of lycopene supplementation. Multiple mechanisms contribute to the effect of tomato products. The antioxidant properties of carotenoids, mainly lycopene, are central to their beneficial properties. The processing of tomatoes and cooking them with olive oil increases the carotenoid bioavailability. Carotenoids prevent the oxidation of LDL-cholesterol, and some studies report a mild LDL-cholesterol reduction effect after tomato juice
supplementation [24]. Moreover, anti-inflammatory benefits have been ascribed to tomato products; however, published data are conflicting [2,25]. Plasma lipid peroxides were measured as a cumulative marker of TOS. The decline in TOS at the end of the supplementation period demonstrates the midterm antioxidant potential of tomato paste. Nevertheless, TOS did not change acutely after ingestion of the first serving. The concurrent TOS reduction and improvement in FMD at day 15 could be explained through a cause-and-effect mechanism. Thus, ingestion of tomato paste abundant in antioxidants could decrease TOS, resulting in a favorable redox state for vessel walls, which would increase the production of vasodilatory molecules (nitric oxide, adenosine). However plausible this scenario is, it cannot be substantiated by the nature of our in vivo study. Confirmation of this mechanism would require ex vivo protocols. We chose to use a commercially available tomato product instead of pharmaceutical preparations of lycopene. This allows for the direct extrapolation of our findings to everyday culinary practices. The amount of lycopene contained in a serving of tomato paste exceeds the average dietary intake; nonetheless, it represents a physiologically relevant dose that compares favorably to doses used in similar human studies in the past [8,10]. The beneficial effect of tomato products may be attributed to an additive/synergistic effect of lycopene with other micronutrients, including α-tocopherol, vitamin C, and rutin [26]. The decision to provide the tomato paste in the morning was based on the pharmacokinetics of lycopene. Tomato products are usually consumed during lunch and/or dinner; heating during cooking increases lycopene bioavailability by releasing it from the food matrix. Concomitant fat consumption may also improve bioavailability because lycopene is lipid soluble. It is plausible that consumption at lunch or dinnertime could have magnified the salutary impact on endothelial function [11]. Some limitations of our study should be acknowledged. Lycopene concentrations were not measured in blood samples because the necessary high-performance liquid chromatography techniques are not available to us. Total oxidative status was not measured for the control arm. A fixed dose of
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400 μg of nitroglycerin was used during NMD measurements. Confirmatory research is also needed to evaluate the role of the dietary portions of tomatoes; we used an amount of lycopene that exceeds the average daily intake. The inclusion of smokers could have blunted the beneficial effects of tomato paste, given the effect of smoking on the antioxidant properties of bioactive compounds. In conclusion, we have demonstrated that a midterm tomato dietary supplementation period improved endothelial function in a group of healthy young men and women. Individuals with lower baseline FMD values were particularly benefited, as demonstrated by the fact that they exhibited greater increases in FMD values after consumption of tomato paste. Further research is warranted to ascertain the impact of long-term tomato supplementation on endothelial function and CVD risk in various clinical settings.
Acknowledgment The study was funded by Athens Medical School, Greece. There are no conflicts of interest to disclose pertaining to this study.
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