Dietary restriction of lycopene for a period of one month resulted in significantly increased biomarkers of oxidative stress and bone resorption in postmenopausal women

Abstract

Background and Objectives

Lycopene is a carotenoid commonly found in tomatoes and tomato products which acts as an antioxidant to decrease oxidative stress and osteoporosis risk. We wanted to determine the effects of a lycopene-restricted diet on oxidative stress parameters and bone turnover markers in postmenopausal women.

Setting

St. Michael's Hospital, Toronto, ON, Canada.

Participants and Study Design

23 healthy postmenopausal women, 50–60 years old, provided blood samples at baseline and following a one-month lycopene-depletion period.

Measurements

Serum samples were analyzed for carotenoids; the oxidative stress parameters protein thiols and thiobarbituric-malondialdehyde reactive substances; the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), and the bone turnover markers bone alkaline phosphatase and crosslinked N-telopeptide of type I collagen (NTx). A paired t-test was used to test for significant differences in bone turnover markers, oxidative stress parameters and antioxidant status after lycopene restriction.

Results

Dietary lycopene restriction resulted in significantly decreased serum lycopene (p<0.0001), lutein/zeaxanthin (p<0.01), and α-/β-carotene (p<0.05). GPx (p<0.01), lipid and protein oxidation increased (not significant), while CAT and SOD were significantly depressed (p<0.05 and p<0.005, respectively). These changes coincided with significantly increased NTx (p<0.05).

Conclusions

These findings suggest that the daily consumption of lycopene may be important as it acts as an antioxidant to decrease bone resorption in postmenopausal women and may therefore be beneficial in reducing the risk of osteoporosis.

Introduction

Lycopene is the most predominant carotenoid found in human serum (1). Over 80% of lycopene consumed in the diet is obtained through consumption of tomatoes and tomato products (2). However, it is also found in watermelon, pink grapefruit, rosehips, and pink guava (3). Lycopene is a 40-carbon, acyclic isomer of ß-carotene. Among the carotenoid family, it is credited with the highest singlet oxygen quenching capacity (4), which makes it a powerful antioxidant. Lycopene contains 11 conjugated and 2 unconjugated double bonds and exists in different isomeric forms. There is the all-trans form of lycopene, and several different cis isomers, which are formed after rotation of any of the 11 conjugated double bonds. Cis isomers are credited with the highest antioxidant capacity, with 5-cis lycopene having the highest antioxidant capacity and all-trans lycopene having the lowest (3). In lycopene-containing foods such as raw tomatoes, lycopene exists primarily in the all-trans form, but in human serum a higher concentration of cis isomers exists (2). This increase in isomer concentrations could result from exposure to high temperatures during food processing but more likely it is a result of selective uptake and/or differential bioavailability of cis isomers (1, 3).

Lycopene is well documented for its ability to decrease biomarkers of oxidative stress. Research shows that it is capable of decreasing several lipid, protein and DNA markers (3). In some studies, it has also demonstrated a capacity to increase markers of antioxidant capacity and endogenous antioxidant enzymes (5, 6).

The antioxidant properties of lycopene have been credited with its ability to decrease the risk of age-related chronic diseases often attributed to oxidative stress. Epidemiological and dietary intervention studies suggest that through its antioxidant capacity, lycopene may decrease the risk of infertility (7, 8), diabetes (9, 10), dementia (11, 12), cardiovascular disease (13, 14) and several types of cancer (15, 16, 17). Our previous study (18) provided evidence that lycopene may also decrease the risk of osteoporosis in postmenopausal women. Many of these intervention studies require a washout period, during which lycopene is restricted in the diet. Dietary lycopene restriction is necessary to obtain a clinically relevant response to lycopene, as it has been shown that higher baseline lycopene concentrations result in a moderate to null response to lycopene (19). However, studies on the effects of a carotenoid-depleted diet are limited and those that exist examined small study populations (N<10), very few biomarkers of oxidative stress, and did not report on how this type of diet would affect the risk of developing age-related chronic diseases (20, 21, 22). Studies on lycopene depletion are important not only because they may delineate crucial functions, but they also help to elucidate mechanisms which may not be accurately demonstrated in intervention studies where absorption saturation occurs (23).

The objectives of the present study were to determine the effects of dietary lycopene restriction in postmenopausal women, and whether refraining from consuming lycopene containing foods for a period of just one month would result in significant effects on oxidative stress parameters, antioxidant capacity and bone turnover markers.

Matrials and methods

Study design, participant recruitment, and blood sample collection

Our protocol was approved by the Research Ethics Board at St. Michael's Hospital, Toronto, Ontario, Canada and followed the guidelines of good clinical practices according to the Declaration of Helsinki. Female participants between 50-60 years old, who were at least one year postmenopausal, were recruited by telephone and advertisements. Any participants who were on medications for heart disease, high blood pressure, diabetes and/or osteoporosis were excluded, as were participants who smoked cigarettes. Participants were instructed to maintain their usual, daily diets, and then submitted dietary records outlining foods, beverages, and nutritional supplements consumed over the previous 7 days and provided a baseline 12-hour fasting blood sample. Participants were given a list of lycopene-containing foods to avoid for the remainder of the study, which included tomatoes and tomato products, vegetable juice, watermelon, Chinese orange, pink guava, red grapefruit, and rosehip. Another set of dietary records and a fasting blood sample was collected following a one-month washout period during which no lycopene containing foods were consumed. Participants who did not show a greater than 20% change in serum lycopene as a result of dietary restriction would be considered non-compliant and would therefore be excluded; no participants were excluded based on this criteria.

Dietary lycopene analyses

Lycopene intake was analyzed using the U.S. Department of Agriculture national nutrient database for lycopene as a reference, which lists the content of lycopene in each food in μg/measure (24). Using this information, the lycopene content was calculated in milligrams for each food, and an average of the total daily lycopene consumed was calculated for each participant at baseline and after dietary lycopene restriction.

Measurements of antioxidant capacity

Unless otherwise specified, all materials were obtained from Sigma Aldrich Canada, Oakville, ON, Canada The concentrations of serum lycopene, lutein/zeaxanthin, and a-/ß-carotene were measured using high performance liquid chromatography (HPLC), according to previously published methods (25), with minor modifications. Serum carotenoids were extracted 3 times using a solution of 0.0625% BHT-Ethanol, 0.005% BHT-Hexane and echinenone internal standard (CaroteNature, Switzerland). HPLC was carried out using the Waters 2690 Alliance HPLC System and a Waters 996 PDA detector (Milford, MA, USA). The carotenoids were analyzed at a wavelength of 450 nm and were determined using external standard calibration curves on the Waters Millennium data management software, 4.0 edition (Milford, MA, USA).

Measurements of oxidative stress parameters and endogenous antioxidant enzymes

Protein oxidation was determined by estimating protein-sulfhydryl groups (thiols) in serum (26). A high concentration of protein thiols corresponds to a lower protein oxidation. Lipid peroxidation was measured in the serum using the thiobarbituric acid-malondialdehyde assay and was reported as TBA reactive substances (TBARS) (27).

Total Hb was detected in RBC hemolysates using the Drabkin's method (Sigma Aldrich, Canada), according to the manufacturer's protocol. CAT was measured using the H2O2 decomposition method (28) and was expressed as K/g Hb, where K = 2.3/(A time)log OD₁₅ seconds/OD soseconds. SOD was determined by the auto-oxidation of epinephrine (29) and enzyme activity (U) was defined as 50% inhibition of the rate of epinephrine oxidation compared to a negative control sample normalized to milligrams of Hb and expressed as U/mg Hb. GPx activity was measured by the decrease in NADPH absorbance (30) and one enzyme unit of GPx was defined as the number of micromoles of NADPH oxidized per minute, where OD1μM₃₄₀ =6.2, normalized to grams Hb and expressed as U/g Hb.

Measurements of bone turnover markers

ELISA was used to measure the serum concentrations of the bone resorption marker crosslinked N-telopeptide of type I collagen (NTx) (INTER MEDICO, Ontario) and the bone formation markers bone-specific alkaline phosphatase (BAP) (ESBE Scientific, Ontario). NTx values were expressed as nanomoles of bone collagen equivalents (nM BCE) and BAP was expressed as U/L, where one Unit represented lμmol of p-nitrophenyl phosphate hydrolyzed per minute at 25°C in 2-amino-2-methyl-l-propanol buffer.

Statistical Analyses

All statistical analyses were performed using GraphPad PRISM^TM 5.00 for Windows (GraphPad Software, California). Summary statistics of participant demographics were generated presented as means ± standard errors of the mean (SEM). A paired t-test was used to test for significant differences in bone turnover markers, oxidative stress parameters and antioxidant status after dietary lycopene restriction. For data that were not normally distributed, the Grubb's test for outliers was used to exclude the offending outlier. If data were not normally distributed after this test, then the Wilcoxon matched pairs test was used. Statistical significance was considered at p<0.05. Percent change in bone turnover markers, oxidative stress parameters, and antioxidant status were also calculated to determine trends of the change. Any other statistics used are specified in the text.

Results

Participant characteristics

Twenty-three postmenopausal women participated in this study. The characteristics for these participants are shown in Table 1. The average lycopene intake for participants prior to the restrictive diet was 3.5 milligrams (mg) per day ± 0.6 (Table 1). The range of lycopene intake was quite wide, from 0 to 12.3 mg per day, with 39% of participants consuming 2 mg or less per day. In North America, approximately 50% of the population consume a relatively low intake of 2 mg or less of lycopene per day (3), however, according to studies by us and others (31, 32) the average lycopene intake in Canadian women is approximately 5-6 mg/day. Considering the fact that the majority of clinical trials supply at least 15 mg/day to determine the effects of lycopene (33), an intake of = 2 mg per day is quite low.

Table 1.

Participant characteristics as determined at the beginning of the study for 23 postmenopausal, female participants, aged 50-60

Baseline participant characteristic	Mean ± SEM
Age (years)	54.4 ± 0.6
BMI (kg/m2)	24.7 ± 0.9
Years since menopause	4.1 ± 0.6
Daily lycopene intake (mg/day)	3.5 ± 0.6
Total serum lycopene (nM)	1171.0 ± 111.1

Dietary lycopene restriction significantly decreased all-trans and total cis serum lycopene concentrations

After one-month during which participants refrained from consuming lycopene-containing foods, mean lycopene intake significantly decreased from 3.50 mg/day ± 0.60 to 0.13 mg/day ± 0.06 (p<0.001). This corresponded to a significant decrease in total serum lycopene (p<0.0001) of 54.9% ± 3.6 (Table 2). Concentrations of both all-trans and cis isomers were significantly lower in the serum of participants after lycopene restriction (p<0.0001) (Figure 1). In fact, all-trans lycopene decreased by an average of 59.0% ± 4.5 (p<0.0001), and total cis lycopene (5-cis and other-cis) decreased by an average of 49.7% ± 3.9 (p<0.0001).

Table 2.

Changes in serum carotenoid concentrations after postmenopausal female participants refrained from consuming lycopene containing foods for a period of 1 month

Carotenoid	Concentration in serum (nM)		Results of paired t-test	Average % change
	Baseline) (mean ± SEM	Lycopene restricted (mean ± SEM)		(mean ± SEM)
-carotene	408.4 ± 131.4	334.4 ± 110.6	p<0.05∗	-13.03 ±6.88
-carotene	1443.0 ± 278.9	1035.0 ±221.7	p<0.0005∗	-22.84 ±5.09
-cryptoxanthin	403.2 ± 58.4	367.6 ± 47.3	p = 0.229	-1.29 ± 8.21
Lycopene	1171.0 ± 111.1	494.9 ± 48.46	p<0.0001	-54.86 ± 3.59•
Lutein/zeaxanthin	516.4 ±49.57	443.0 ± 47.04	p<0.01	-12.77 ± 5.03

^∗Wilcoxon matched pairs test used for these non-normally distributed data sets. • Average percent change in lycopene was significantly higher than that seen for all of the other carotenoids (p<0.0001), as determined by unpaired t-test or Mann-Whitney test.

Figure 1.

Serum concentrations of all-trans and total cis lycopene in 23 postmenopausal female participants who refrained from consuming lycopene-containing foods for a period of one month. Values are mean ± SEM; baseline and lycopene restricted values were compared using a paired t-test (a p<0.0001)

In addition to significant decreases of 54.9% ± 3.6 in serum lycopene, there were also significant decreases of 12.8 %± 5.0 for serum lutein/zeaxanthin (p<0.01), 13.0% ± 6.9 for serum a-carotene (p<0.05), and 22.8% ± 5.1 for ß-carotene (p<0.0005) after dietary lycopene restriction (Table 2) compared to baseline values. However, the overall percent change in these serum carotenoids was not as high as that seen for lycopene. In fact, an unpaired t-test was performed and the percent change in lycopene (-54.9%) was significantly greater than that of each of the carotenoids (p<0.0001 for serum lycopene vs. each carotenoid), demonstrating the greatest decrease in serum carotenoids after restriction was seen for lycopene.

Dietary lycopene restriction increased oxidative stress parameters and affected endogenous antioxidant enzyme activities

Lycopene restriction for a period of one month resulted in marginally decreased protein thiols from 423.7mM ± 19.31 to 392.3 mM ± 14.22, conferring an apparent increase in protein oxidation (not significant, p=0.05). There was also a marginal increase in TBARS, from a concentration of 8.08 nmol/ml ± 0.44 to 9.18 nmol/ml ± 0.76, indicating an apparent increase in lipid peroxidation (not significant, p<0.10). Parameters of protein oxidation and lipid peroxidation increased by 5.5% ± 3.3 and 14.5% ± 7.1, respectively.

Concentrations of the endogenous antioxidant enzymes CAT and SOD were significantly decreased after lycopene restriction (p<0.05, and p<0.005, respectively) (Figure 2). The average decrease was 8.4% ± 9.3 for CAT and 22.7% ± 11.8 for SOD. Conversely, concentrations of GPx were significantly increased (p<0.01) (Figure 2) after lycopene restriction with an average increase of 114.7% ± 35.8 in enzyme activity. There was a significant, positive linear relationship between this change in GPx and the change in TBARS after lycopene restriction (slope = 0.050, p<0.05, data not shown).

Figure 2.

Enzyme activity of the endogenous antioxidant enzymes, CAT, SOD, and GPx, at baseline and after 1 month of lycopene restriction in 23 postmenopausal participants. Values are mean ± SEM; baseline and lycopene restricted values were compared using a paired t-test (a p<0.05, b p<0.005, c p<0.01, respectively)

Dietary lycopene restriction significantly increased the bone resorption marker NTx

Postmenopausal participants who refrained from consuming lycopene for a period of 1 month had significantly increased concentrations of the bone resorption marker, NTx (p<0.05) (Figure 3). The average increase in NTx was 20.6% ± 9.8. The bone formation marker, BAP, remained stable throughout the study period and there were no changes as a result of lycopene restriction (data not shown).

Figure 3.

Concentrations of the bone resorption marker, NTx, in 23 postmenopausal participants who refrained from lycopene consumption for a period of 1 month. Values are mean ± SEM; baseline and lycopene restricted values were compared using a paired t-test (a p<0.05)

Discussion

This paper presented data showing that dietary lycopene restriction, for a period of only one month, resulted in important changes in biomarkers of oxidative stress and bone resorption markers. Refraining from consuming lycopene-containing foods resulted in significantly lower serum carotenoids, particularly lycopene, which coincided with apparent increases in oxidative stress parameters, significant increases in the bone resorption marker NTx and GPx enzyme activity, and depressed CAT and SOD enzyme activities. To our knowledge, this is the first study reporting on the effects of dietary lycopene restriction on these parameters in postmenopausal women who are at a high risk for osteoporosis.

The average lycopene intake at baseline for this subset of participants was lower, at 3.5 mg per day, compared to the 6.23 mg/day reported by us (p<0.05) (32) and the 5.26 mg/day reported by others (31) for Canadian women in this age group. The fact that participants in this study, with an initial lower than average lycopene intake, demonstrated such significant and important changes in biomarkers of oxidative stress biomarkers and bone resorption after only one month of refraining from consuming lycopene-containing foods, further illustrates that even a small daily intake of lycopene is biologically important and may offer protection against the effects of oxidative stress, particularly on bones.

The reported half-life of lycopene in human serum ranges from as little as 2-3 days (34), to as high as 12-33 days (35). Restriction of lycopene-containing foods for only 12-14 days has been reported to decrease serum lycopene by 50%, which is similar to what we have reported here after 30 days (Table 2). This change in serum lycopene after restriction, reported by us and others (35), suggests that the simultaneous effects seen here on biomarkers of oxidative stress and bone resorption occur as a direct result of lycopene depletion.

The apparent changes seen on oxidative stress biomarkers, antioxidant enzymes and bone resorption may not be solely due to a lack of lycopene. While lycopene accounts for greater than 85% of the carotenoids found in tomatoes and tomato products (3), these foods do contain modest concentrations of other carotenoids, such as ß-carotene (24). Supplementation with tomato products has been shown to significantly increase not only lycopene but other carotenoids (36), and these carotenoids have been shown to work in synergy to exert beneficial effects (37). We reported significant decreases in not only lycopene, but lutein/zeaxanthin and a- and ß-carotene, which is consistent with other studies on carotenoid depletion (38). Although the change in serum lycopene in our participants was significantly higher than the change seen in the other carotenoids after depletion (p<0.0001), it is entirely possible that there was an effect of restriction of other carotenoids present in tomatoes and tomato products on the parameters studied.

After 30 days of dietary lycopene restriction, there were significant increases in the bone resorption marker NTx (p<0.05), corresponding to an increase of 20.6% ± 9.8. This significant increase in the bone resorption marker NTx may lead to a long-term decrease in BMD and increased fracture risk (39), suggesting that a longer restriction period may be detrimental to bone health, particularly in this group of postmenopausal women who were already at higher risk for osteoporosis. It is for this reason that a longer period of dietary lycopene depletion for the purpose of clinical intervention trials, particularly in postmenopausal women, should be avoided.

It was to be expected that antioxidant enzyme concentrations would decrease after dietary lycopene restriction, since it has been suggested that consumption of antioxidants, particularly lycopene, may increase the activities of these endogenous enzymes (6, 20). It has been reported that 68 days of a carotenoid depleted diet resulted in significantly lower SOD activity (20). In the present study, the antioxidant enzymes may act to decrease oxidative stress resulting from dietary lycopene as a compensatory mechanism, and are thus decremented in the process. This explains the depressed levels of CAT and SOD shown in these participants. However, GPx activity significantly increased as a result of dietary lycopene restriction (Figures 2). A similar finding on GPx is reported in a study on ß-carotene depletion in premenopausal women (22). GPx is involved in the reduction of lipid hydroperoxides, a by-product of lipid peroxidation, into non-detrimental alcohols (40). This role of GPx in reducing lipid peroxidation, suggests that the increased GPx shown after lycopene restriction may be due to the concomitant increase in lipid peroxidation. In fact, there is a significant, positive, association between change in GPx activity and TBARS (p<0.05), indicating that the increase in GPx was indeed to protect against the increasing lipid peroxidation which occurs after lycopene restriction, as previously reported (41). The compensatory mechanisms of CAT, SOD, and GPx associated with a lycopene-deprived diet may be the reason why the increase in oxidative stress was not significant. It is possible that these antioxidant enzymes acted in place of lycopene, moderating the resultant increases in oxidative stress biomarkers. A longer period of depletion may have magnified the reported effect of dietary lycopene restriction on lipid and protein oxidation (20, 21).

The implications of the findings reported here are important and may assist in establishing guidelines for future clinical trials in which a period of dietary lycopene restriction is required, by yielding information on the effects of dietary restriction, particularly on bone health. In addition, lycopene is present in a select number of foods; therefore not consuming these products as a part of the regular daily diet may result in negative health consequences in women, particularly with respect to bone health. Results from this study suggest that the consumption of tomatoes and tomato products, as a source of lycopene in the daily diet, may be beneficial in maintaining overall health and decreasing the risk for age-related chronic diseases, particularly osteoporosis, which is associated with oxidative stress.

Acknowledgements: Funding is shared by the Canadian Institutes of Health Research (CIHR) and the Research and Development Departments of Genuine Health Inc., the H.J. Heinz Co, Millenium Biologix Inc. (Canada), Kagome Co. (Japan), and LycoRed, Ltd. (Israel). We especially thank Dr. R.G Josse, Dr. C. Derzko, and A. Strauss for their contributions to participant recruitment.

Conflict of interest statement: E.S. Mackinnon, A.V. Rao and L.G. Rao have no conflicts of interest regarding this manuscript.

Statement of authorship: The author's responsibilities were as follows: ESM participated in study design, carried out the study, data analysis, and drafted the manuscript, AVR and LGR conceived of the study, participated in its design and coordination, and helped to draft the manuscript, LGR was the Principal Investigator of the project.