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2 Colorado State University, Fort Collins, CO 80523 and 3 AMC Cancer Research Center, Denver, CO 80214
* To whom correspondence should be addressed. E-mail: henry.thompson{at}colostate.edu.
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ABSTRACT |
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 TOP ABSTRACT IntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Introduction |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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The DBC in plant foods are likely to modulate the activity of many biological systems in mammalian species (1). However, there is little question that the most widely investigated effect is the role that phytochemical DBC play in protecting biological systems against cellular oxidation. In an effort to extend studies of phytochemical antioxidants from their assessment in test tube assays, cell culture systems, and preclinical models to clinical interventions, our laboratory reported a series of investigations of the effects of carefully formulated diets that varied in their VF content (6–8). In those studies, high (>10 servings of VF/d) vs. low (<3.5 servings of VF/d) intake was associated with a modest reduction in both the level of DNA oxidation in peripheral lymphocytes and the concentration of a marker of lipid peroxidation excreted in urine. Antioxidant effects were also manifest primarily in individuals with elevated levels of oxidation biomarkers at the time they initiated participation in an experiment. These findings provided the foundation for the study reported herein; the primary question asked was whether the botanical diversity of the plant food component of the diet alters the response in oxidative biomarkers to high VF consumption.
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Materials and Methods |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Volunteers for this study were recruited via program advertisement among a group of individuals interested in women's health issues. The eligibility criteria for participation were as follows: female, 
21 y old, not pregnant or lactating, no known food allergies, not consuming a special diet prescribed by a physician, familiar with cooking, willing to prepare prescribed meals for 2 wk, willing to keep a record of the foods eaten during the study, regularly consumes 
2 alcoholic beverages/d, does not use tobacco products of any type, does not take multivitamin supplements or willing to stop taking vitamin supplements while participating in the study, not a competitive athlete, maintained approximately the same body weight for the past 6 mo, and does not regularly take medications for things other than birth control or postmenopausal hormone therapy. Failure of interested individuals to meet eligibility criteria formed the basis for exclusion from the study. The clinical protocol was approved by the Institutional Committee for the Protection of Human Subjects at the AMC Cancer Research Center. A total of 111 women enrolled in the study and gave informed consent.
Dietary approach
To provide a systematic framework for addressing the question concerning whether the type of plant foods comprising the diet affects antioxidant activity in vivo, plant foods were categorized by botanical family. Using this approach, 2 diets were formulated to provide 8–10 servings of VF/d; one had low botanical diversity (LBD), whereas the second had high botanical diversity (HBD). The HBD diet was formulated from the same 18 botanical families that we investigated previously, families that were shown to induce a modest reduction in oxidative damage of lipids or DNA (8). Both diets were designed to provide 8–10 servings of VF/d depending on the participant's energy intake. The serving size definitions are those adapted by the 5-A-Day for Better Health program (9). Each diet consisted of a fully defined 14-d menu of recipes. Both diets were designed to have the same macronutrient content (% energy from fat, 30%; protein, 18%; carbohydrate, 52%), to meet the Recommended Dietary Allowances for vitamins and minerals, and to be balanced in nutrients from the various food groups.(10)
Study design
Based on our previous work in which we found no evidence that effects of VF observed after 2 wk changed over the next 6 wk of treatment (7), a 2-wk duration for the intervention was chosen for this work. Women who met the eligibility criteria and gave informed consent were randomly assigned to either the HBD or LBD diet groups. Blood samples (from nonfasting subjects) were taken in the morning on which the intervention was initiated, and again on the morning after the last day of the dietary intervention. First, void urine specimens were collected on 3 consecutive mornings preceding each blood draw. The procedure for, and timing of sample collection were based on previous work (6). A total of 111 individuals were randomized, and 53 participants in each diet group completed the study.
Laboratory Measurements
Urinary 8-iso-PGF2
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Analysis of 8-isoprostane F2 (8-iso-PGF2) in urine provides a time-averaged index of lipid peroxidation over a relatively prolonged interval that offers greater utility than the more transitory information provided by analysis of blood. Moreover, urine contains very little arachidonic acid, thereby limiting artifactual oxidation and isoprostane production after sample collection. Because we found that intra-individual daily excretion of urinary 8-iso-PGF2 varies considerably in human subjects, we elected to measure 8-iso-PGF2 abundance in pooled urine samples generated from multiple specimens. Urine from the first void of the morning was collected without preservative in plastic vessels on 3 consecutive days. The decision to use first voids rather than 24-h collections was based on our pilot data indicating that 24-h means expressed/mg creatinine did not differ from values obtained from first voids and also on our experience that collecting reliable 24-h urine samples from free-living subjects is problematic. After solid phase extraction of the urine, the analysis was performed using an ELISA assay (Cayman Chemical); our method was described previously in detail (7).
Lymphocyte 8-oxo-dG. To evaluate the effects of the 2 diets on DNA oxidation, the concentration of 8-hydroxy-2-deoxyguanosine (8-oxo-dG) in DNA isolated from peripheral lymphocytes was determined. 8-oxo-dG is a relatively abundant and readily detected product of oxidative DNA damage; as such, it is regarded as a useful and relevant marker for cellular oxidative stress (11). The decision to assess oxidative damage in DNA isolated from lymphocytes was made for several reasons. Lymphocytes are relatively easy to obtain, their half-life in blood is short, and they provide a general estimate of oxidative stress, at the genomic level, to which an organism is subjected (12,13). Our HPLC method using electrochemical detection was described in detail in a recent publication (8).
Plasma carotenoids.
Plasma concentrations of -carotene, ß-carotene, lycopene, lutein, and ß-cryptoxanthine were determined to provide a biochemical index of compliance with the intervention diets. Our HPLC method using diode array detection was described in detail (7).
Other measurements. At baseline, participants completed the NIH 7-item screening questionnaire for fruit and vegetable intake (14); this was the only assessment of preintervention dietary behaviors that was conducted. Participants also completed printed surveys at baseline and postintervention. The baseline surveys provided data on demographics and levels of physical activity. The postintervention surveys provided self-reported compliance and feedback on the intervention.
Statistical analyses
Differences in categorical variables at baseline across randomization groups were evaluated using Fisher's exact test for independence of proportions (15). Continuous data were tested for differences in means using a 2-group, 2-sided t test. Differences with a P-value 0.05 were considered significant. Distributions for the continuous data were also checked to determine whether transformations were appropriate. None were deemed necessary in this sample.
Maximum likelihood estimates of a repeated-measures model were used for the analysis of the primary outcomes (concentrations of lymphocyte 8-oxodG and urinary 8-iso-PGF2) (16). This approach is conceptually identical to multivariate ANOVA, but avoids the case-wise deletion of subjects with missing assessments. The model provides unbiased estimates under the less restrictive assumption that missing data are missing at random. The maximum likelihood estimates are based on a repeated-measures model of time x group:
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where yij is the outcome measure for the ith subject in the jth randomization group; j = {1,2}; t = 11; G = A if the subject is in the HBD diet group, B otherwise. Hypotheses were tested by setting up appropriate contrasts (linear combinations of model parameters) within the model; we report the P-values associated with the resulting t statistics. A similar model was used to estimate group differences by quartile of the baseline measure; parameterization was modified to facilitate the contrasts. No statistical adjustments for multiple comparisons and multiple end points were made, but the quartile analysis is considered secondary and results should be interpreted with caution. All analyses were done using SAS v 8.2 (SAS Institute).
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Results |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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- and ß-carotene, lutein, lycopene, and ß-cryptoxanthin, were evaluated as markers of compliance with the dietary intervention. Based on participants' daily food logs, intake of dietary carotenoids was markedly and significantly different between the LBD and HBD diet groups (Table 3). In both intervention groups, changes in plasma carotenoid concentrations paralleled the intake of dietary carotenoids reported in Table 4. The plasma carotenoid data were consistent with the high level of self-reported compliance.
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The urinary concentration of 8-iso-PGF2 was lower in the postintervention than in the preintervention samples of subjects in both intervention groups (Table 5, P < 0.001). The extent of reduction tended to be greater in the HBD intervention group, although the postintervention concentrations of 8-iso-PGF2 in the HBD and LBD diet groups did not differ significantly (P = 0.141).
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is related to the level of this analyte in the urine at baseline. To examine the effect of botanical diversity on lipid peroxidation, as affected by peroxidation status at baseline, the data were subdivided by baseline quartile of urinary 8-iso-PGF2 and the effects of the dietary interventions evaluated. Neither intervention diet affected the concentration of 8-iso-PGF2 in individuals in the lowest quartile. The HBD intervention significantly reduced the urinary concentration of 8-iso-PGF2 in the 2nd through 4th quartiles, whereas the effect of the LBD diet was significant only for individuals in the 3rd and 4th quartiles. Lymphocyte 8-oxo-dG. The concentration of 8-oxo-dG in genomic DNA isolated from peripheral lymphocytes (Table 6) was lower in the postintervention than in the preintervention samples from subjects in the HBD diet group (P = 0.05), but not the LBD diet group (P = 0.78). These data were further evaluated by dividing the participants into quartiles by concentration of lymphocyte 8-oxo-dG at baseline. The LBD diet did not affect the concentration of this analyte irrespective of the baseline concentration of 8-oxo-dG. On the other hand, in the HBD, the concentration of 8-oxo-dG was lower in post- than in preintervention samples of participants in the 3rd (P = 0.003) and 4th quartiles (P = 0.007).
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Discussion |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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were reduced to a greater extent by the HBD diet than by the LBD diet. Detailed consideration of these findings is presented below.
There was a significant decrease in the urinary concentration of 8-iso-PGF2 in response to either the LBD or HBD diet (Table 5). Although the effect was greater in women that consumed the HBD diet, that difference was not significant (LBD vs. HBD, P = 0.141). This finding was contrary to our initial prediction that the reduction in oxidation would be greater in the LBD group because that diet was formulated primarily from botanical families considered to be particularly rich sources of foods with high potential for antioxidants effects. The fact that a reduction in oxidative damage to DNA (Table 6) was observed only in the HBD diet group provides additional evidence that the LBD and HBD diets differed in their in vivo activity in reducing cellular oxidation.
Our laboratory recently published evidence supporting the hypothesis that the response to dietary antioxidants is not uniform, but rather is conditional, based on a participant's oxidation status at the time the participant enters a study (7,8). Hence, null effects of a potential antioxidant intervention could be due to the oxidation status of the study population rather than the lack of antioxidant activity of the intervention. To determine whether this might account for our observations, the 8-iso-PGF2 and 8-oxo-dG data were subjected to subgroup analysis based on preintervention concentrations of each analyte. Those analyses indicate that the reduction of lipid peroxidation was observed primarily in the participants in the highest 2 quartiles of baseline 8-iso-PGF2, an observation consistent with our previous reports. Interestingly, both the quartile in which the HBD diet was observed to reduce lipid peroxidation significantly, and the magnitude of the reduction observed in each quartile relative to the effects of the LBD diet, are consistent with the HBD diet having greater activity in reducing lipid peroxidation than the LBD diet. The results of a quartile-based analysis of the effects of the 2 diets on concentrations of 8-oxo-dG in genomic DNA isolated from peripheral lymphocytes (Table 6) also support the hypothesis that only individuals with high baseline levels (3rd and 4th quartiles) of 8-oxo-dG experienced a reduction in the concentration of this analyte, and this effect was significant only in individuals consuming the HBD diet. Hence, these results are also consistent with the existence of a differential response in oxidative damage to the LBD vs. HBD diet.
It is likely that the LBD and HBD diets provided different amounts and types of phytochemicals, an observation supported by the carotenoid intake profiles for each diet (Table 3). Although differences in the chemical composition of these diets undoubtedly contributed to the effects on the peroxidation of lipids and oxidation of DNA, speculation about what the key chemical factors might be is considered unwarranted because so many other issues relating to absorption and metabolism of ingested phytochemicals could also contribute to the observed effects. On the other hand, what can be learned from these data is that the botanical diversity of the diet is likely to play a role in determining the bioactivity of dietary chemicals and that smaller amounts of many phytochemicals may have greater potential to exert beneficial effects than larger amounts of fewer phytochemicals. If this observation is correct, it gives added meaning to one of the most widely pronounced dietary recommendations, i.e., that variety and moderation are the key to a healthy diet.
A growing number of studies of the effects of plant foods, their DBC, and of known antioxidants on oxidative cellular damage in vivo are being reported, and the results are mixed. The significant number of null findings has spawned the antioxidant conundrum as outlined by Seifried et al. (20). The contribution made by the botanical diversity of the diets studied in explaining the mixed results was not investigated previously. The findings presented here represent an initial inquiry concerning the potential importance of plant food selection in a high VF background in determining biological activity; they demonstrate an approach that makes it feasible to investigate the effects of botanical diversity on disease risk biomarkers in a systematic manner.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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4 Abbreviations used: DBC, dietary bioactive compounds; HBD, high botanical diversity; 8-iso-PGF2, 8-isoprostane F2; LBD, low botanical diversity; 8-oxo-dG,8-hydroxy-2-deoxyguanosine; VF, vegetables and fruit.
Manuscript received 3 January 2006. Initial review completed 13 February 2006. Revision accepted 4 May 2006.
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LITERATURE CITED |
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