Effects of docosahexaenoic acid supplementation on blood lipids, estrogen metabolism, and in vivo oxidative stress in postmenopausal vegetarian women

Abstract

Background:

Vegetarians are generally deficient in long-chain n-3 fatty acids. Long-chain n-3 fatty acids have a beneficial effect on plasma lipid levels, and some studies showed that they had breast cancer suppression effect. One of the biomarkers of breast cancer risk is the ratio of urinary 2-hydroxyestrone (2-OHE1) to 16α-hydroxyestrone (16α-OHE1).

Objective:

To investigate the effect of docosahexaenoic acid (DHA, 22:6n-3) supplementation on blood lipids, estrogen metabolism and oxidative stress in vegetarians.

Design:

Single-blind, randomized, placebo-controlled trial.

Interventions:

Twenty-seven postmenopausal vegetarian women were recruited. After a 2-week run-in period with 6 g placebo corn oil, the subjects were subsequently randomized to receive either 6 g corn oil (n=13) or 6 g DHA-rich algae oil (2.14 g of DHA/day) (n=14) for 6 weeks. Two subjects in corn oil group withdrew before completion.

Main outcome measures:

Plasma lipids, urinary 2-OHE1 and 16α-OHE1, urinary F2-isoprostanes and plasma α-tocopherol.

Results:

Plasma LDL-DHA and EPA level increased significantly by DHA supplementation. DHA decreased plasma cholesterol (C) levels (P=0.04), but did not influence the levels of plasma TG, LDL-C and HDL-C, α-tocopherol, urinary F2-isoprostanes, 2-OHE1, 16α-OHE1 and ratio of 2-OHE1 to 16α-OHE1 as compared to corn oil.

Conclusion:

DHA supplementation at a dose of 2.14 g/day for 42 days decreases plasma cholesterol but neither does it show beneficial effects on estrogen metabolism, nor does it induce deleterious effects on the observed in vivo antioxidant or oxidative stress marker in postmenopausal vegetarian women.

Sponsorship:

A grant (# DOH89-TD-1062) from Department of Health, Executive Yuan, Taiwan.

Introduction

Vegetarians have a lower status of long-chain n-3 fatty acid (Lee et al., 2000), principally eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), because of the exclusion of fish from their diet. In addition, the high intake of linoleic acid (18:2n-6) from vegetable oils (Lu et al., 2000b) competes with the conversion of α-linoleic acid (18:3n-3) to EPA and DHA. Long-chain n-3 fatty acids have been found to have beneficial effects on the regulation of plasma lipid levels and cardiovascular function (Mori and Beilin, 2001). Higher levels of platelet aggregation were found in vegetarians when compared to their omnivorous counterparts (Li et al., 1999), and long-chain n-3 fatty acid supplementation has been shown to reduce platelet aggregation (Mezzano et al., 2000). DHA supplementation also induced a moderate reduction in the total or LDL-cholesterol:HDL-cholesterol ratio and a slight reduction in triglyceride concentration in vegetarians (Conquer and Holub, 1996). These observations suggest the deficit in long-chain n-3 fatty acid may cause adverse effect in vegetarians.

Long-chain n-3 fatty acids have been consistently shown to inhibit the proliferation of breast cancer cells in vitro (Bernard-Gallon et al., 2002) and in animal models (Cave, 1997). However, results of epidemiological studies examining the association of breast cancer risk and fish or marine n-3 long-chain fatty acids consumption were not entirely consistent (Terry et al., 2003). One of the biomarkers of breast cancer risk is the ratio of urinary 2-hydroxyestrone (2-OHE1) to 16α-hydroxyestrone (16α-OHE1). The two major estrogen metabolites are 2-OHE1 and 16α-OHE1; the former is not estrogenic, whereas the latter is estrogenic and genotoxic (Service, 1998). The proposal of using the ratio of urinary 2-OHE1 to 16α-OHE1 as a breast cancer risk marker is suggested by some (Kabat et al., 1997; Muti et al., 2000) but not all studies (Ursin et al., 2001). Although estrogen is a major risk factor for breast cancer, its level is hardly altered by diet intervention. On the other hand, the pathways of estrogen metabolism can be manipulated by several dietary factors (Michnovicz et al., 1997; Brooks et al. 2004) and perhaps could be better used for fine-tuning breast cancer risk within an ethnic group. The oxidative metabolism of estrogen is catalyzed predominantly by hepatic cytochrome P450 (CYP450) (Huang et al., 1998; Yamazaki et al., 1998). Dietary fish oil increased the concentration of hepatic overall CYP450 and some subtypes of CYP450 in rats (Valdes et al., 1995; Chen et al., 2003). A preliminary study of Osborne et al. (1998) indicated a decreased extent of 16α-hydroxylation of estradiol in women after supplemented with n-3 fatty acids. Vegetarians have been found to have significantly lower risk of some cancers (Fraser, 1999) but not breast cancer (Fraser, 1999; Dos Santos Silva et al., 2002). Therefore, in this study, an attempt was made to determine if estrogen metabolism was modulated to a less carcinogenic pathway after the improvement of long-chain n-3 fatty acid status by algae oil in postmenopausal vegetarian women.

Higher number of double bonds present in n-3 long-chain fatty acids has been postulated to enhance lipid peroxidation. Nevertheless, the influence of fish oil supplementation on some indicators of oxidative stress was not consistent (Meydani et al., 1991; Higdon et al., 2000), partly because n-3 fatty acids also enhanced the activities and expression of antioxidant enzymes, such as catalase, glutathione peroxidase and superoxide dismutase (Takahashi et al., 2002; Wang et al., 2004). Therefore, to examine whether DHA supplementation actually induces oxidative stress in vivo in vegetarians, levels of urinary F2-isoprostanes and plasma α-tocopherol were measured. In addition, LDL oxidation in vitro was also assessed in this study.

Materials and methods

Supplements

DHA-rich oil extracted from algae was used because fish oil was not acceptable to vegetarians. The DHA oil was kindly provided by Westar Nutrition Corp, CA, USA. It was free of EPA and with the addition of 1 IU dl-α-tocopheryl acetate per gram. Corn oil was used as a placebo oil because it was devoid of n-3 fatty acids and was one of the popular cooking oils used by the subjects recruited. Antioxidants were not added to the corn oil and α- and γ-tocopherol contents were natural amounts found in corn oil. Fatty acid composition analyzed in our laboratory and tocopherol contents of DHA oil and placebo corn oil based on manufacturer's specification are shown in Table 1.

Table 1 Fatty acid composition and tocopherol contents of oils

Subjects

A total of 27 postmenopausal vegans and lacto-ovovegetarians were recruited from the local vegetarian societies and religious groups. The inclusion criteria were as follows: (1) having followed the vegetarian diet at least for 1 year; (2) amenorrheic at least for 1 year; (3) age under 60 years; (4) body mass index in the range of 18–26 kg/m2; (5) no history of cardiovascular, metabolic or endocrinologic disease, as evidenced by blood chemistry screening and personal interview; (6) no use of HRT at least for 6 months. The characteristics of the subjects studied are described in Table 2. None of them were nuns. All subjects received single-blinded corn oil placebo during a 2-week run-in period and were subsequently randomized by random digit table to receive either 6 g placebo or 6 g DHA-rich algae oil (2.14 g of DHA/day) per day for another 6 weeks. The amount of n-3 fatty acids ingested per day was approximately equal to that contained in 80 g of salmon (16 g fat/100 g salmon, 16 g n-3 fatty acids/100 g total fatty acids). The odor and taste of algae oil were very mild. The subjects had never known or eaten algae oil before entering this trial, and thus were not expected to distinguish corn oil from algae oil. The subjects were advised to maintain their usual life styles, levels of physical activity, diet habits, and to keep their body weights unvaried. They met technicians to pick up the oils and had body weight measured each week. They were instructed to keep the oil in the refrigerator. Fasting blood and first morning urine samples were collected on the first morning of intervention and the morning just following the end of intervention. Written informed consent was obtained from each participant before inclusion in the study. The protocol was approved by the Human Experimentation Committee of Taiwan Adventist Hospital, Taipei, Taiwan. Two subjects assigned to control group withdrew from the study before completion: one for family problem and the other for an unreported reason.

Table 2 Basic characteristics of the subjects

Collection and analysis of blood and urine samples

After a 12-h fast, blood was collected from subjects into tubes containing EDTA (2.8 mg/ml of blood). Plasma was separated from whole blood by centrifugation at 2000 g for 15 min and stored at −70°C until the end of the study where aliquots from each subjects were analyzed at the same run. Low-density lipoprotein (LDL) and high-density lipoprotein (HDL) were isolated from plasma by sequential ultracentrifugation in NaBr density solution containing 10 μmol EDTA/l at densities of 1.019–1.063, and 1.063–1.210, respectively. The isolated LDL were dialyzed overnight and then diluted to 200 mg protein/l. LDL oxidation was initiated by the addition of CuSO4 to a final concentration of 5 μmol/l for the measurements of thiobarbituric acid reactive substances (TBARS) produced after 3 h oxidation as previously described (Lu et al., 2000b). Fatty acid compositions of corn oil, algae oil and LDL were analyzed according to the method of Lepage and Roy (1986). Plasma cholesterol (C), LDL-C, HDL-C and plasma triglycerides (TG) were measured by using enzymatic kits (Randox Lab., Antrim, UK). Plasma α-tocopherol was determined by HPLC according to the method described by Kaplan et al. (1987).

Early morning spot urine samples were collected into a tube containing vitamin C (1 mg/ml of urine) and immediately cooled to 4°C. After centrifugation at 3000 g for 15 min, the resulting clear supernatants were stored at −70°C until the end of the study where aliquots from each subjects were analyzed at the same time. Urinary creatinine was determined with the help of a commercial kit (Randox Lab. Antrim, UK) after heating at 100°C for 5 min to destroy residual vitamin C. Urinary 2-OHE1 and 16α-OHE1 were measured in triplicate using a competitive solid-phase enzyme immunoassay (EIA) kit (Immuna Care Corporation, Bethlehem, PA, USA) (Bradlow et al., 1998). The monoclonal antibody to 2-OHE1 had a 100% crossreactivity with 2-hydroxyestradiol (2-OHE2), and, thus, the results shown are actually the sum of 2-hydroxyestrogens. Since the level of 2-OHE2 is much less than 2-OHE1, the values were predominantly contributed by 2-OHE1. The samples from the same person were analyzed sequentially in random order in the same plate and each plate included equal number of samples from each group to decrease the artificial variations arising from sample location and plate to plate difference. The kinetics of the immunoreaction was monitored at 405 nm at 2-min intervals for 20 min, using a MR5000 plate reader (Dynatech Lab., VI, USA). The within-plate coefficients of variation were 3.2–10 and 0.9–5.2% and between-plate coefficients of variation were 7.7 and 3.1% for 2-OHE1 and 16α-OHE1, respectively. The results are expressed as ng/mg creatinine to account for differences arising from variations in urine concentration.

Urinary concentration of an F2-isoprostane, 8-iso-prostaglandin F2α was measured by competitive solid-phase EIA kits (Assay Designs, Ann Arbor, MI, USA) and the results are expressed as ng/mg creatinine.

Statistical analysis

Results were expressed in terms of unadjusted means and standard deviations. The normality of data was checked by Kolmogorov–Smirnov test. Normally distributed data, either original or after transformation, were tested by analysis of covariance (ANCOVA) with adjustment for baseline values. Because the unstandardized residuals of log-transformed data for LDL-EPA were not normally distributed, data of LDL-EPA were analyzed by nonparametric test (Mann–Whitney test) to compare changes from baseline between two groups. Results were considered statistically significant at P<0.05. All statistical analyses were conducted by using SPSS 11.5.

Results

Compliance of the subjects was monitored by means of a self-reported daily consumption sheet and confirmed by the changes in LDL fatty acid composition following DHA supplementation. DHA oil supplementation significantly increased the mean levels of LDL-DHA and eicosapentaenoic acid (EPA) (Table 3), but did not change that of arachidonic acid (AA) (Table 3). None of the LDL-fatty acid levels changed significantly after placebo corn oil supplementation (Table 3).

Table 3 Fatty acid composition of LDLa

Plasma cholesterol level had a small but significant decrease after DHA supplementation as compared to corn oil supplementation. Levels of plasma TG, LDL-C, HDL-C, urinary 2-OHE1, 16α-OHE1 and the ratio of 2-OHE1 to 16α-OHE1 did not show a significant difference between two groups after oil supplementations (Table 4). α-Tocopherol status expressed as plasma levels or levels normalized by the sum of plasma TG and cholesterol, and urinary F2-isoprostane did not have significant difference between two groups after interventions (Table 4). TBARS production in in vitro oxidized LDL increased significantly after DHA supplementation (Table 4).

Table 4 Levels of plasma lipids, urinary estrogen metabolites, oxidative stress and antioxidant status of subjectsa

Discussion

Fatty acid status

The n-3 fatty acids supplemented in our study was DHA-rich algae oil, which was devoid of EPA. Although a small portion of DHA could be retroconverted to EPA (Nelson et al., 1997), the conversion is limited. Our study showed that DHA supplementation increased LDL-DHA by 175% and LDL-EPA by 39% (Table 3). LDL-fatty acid compositions might not closely reflect fatty acid status as plasma or erythrocyte phospholipids did (Dougherty et al., 1987). But serum cholesteryl ester fatty acid composition has also been widely used as a biomarker for fatty acid intake (Zock et al., 1997). The fatty acids in LDL are predominantly esterified to cholesterol and they contribute to the majority of plasma cholesteryl ester fatty acids. As a consequence, LDL-fatty acid composition was used as a surrogate index of fatty acid status in this study.

Estrogen metabolites

Fish oil influenced the activity of hepatic CYP450 (Valdes et al., 1995; Chen et al., 2003), which might consequently influence the metabolism of estrogen. The lower status of n-3 long-chain fatty acids in vegetarians seems to be a good model to investigate the influence of DHA supplementation on estrogen metabolism. However, our study did not find significant changes in 2- and 16α-OHE1 after DHA or placebo supplementation (Table 4). In a preliminary study, Osborne et al. (1988) and Karmali (1989) described that women supplemented with fish oil (1.53 g EPA+1.44 g DHA per day) decreased the extent of 16α-hydroxylation of estradiol and women with higher baseline values of 16α-hydroxylation had a more profound decrease. Most of the women in the study were already with an increased risk for breast cancer, whereas those in our study were apparently healthy postmenopausal vegetarians who were not considered to have high breast cancer risk. It is probably hard to further modulate the oxidative metabolism of estrogen in these subjects. The effect of EPA and DHA on estrogen metabolism might be different and needs further investigation. The 6-week duration of intervention was short in this study; however, the metabolic pathway of estrogens was found to be altered in 7 days by indole-3-carbinol (Michnovicz et al., 1997), 12 days by broccoli diet (Kall et al., 1996), 4 weeks or 6 weeks by soy (Lu et al., 2000a; Nettleton et al., 2005). Therefore, 6-week intervention in this study might be adequate. Nevertheless, the small number of subjects and large standard deviations for the estrogen metabolites could have limited the power to examine this effect.

Blood lipids

We only observed a small but significant decrease in plasma cholesterol level (P=0.040), but not in plasma LDL- and HDL-C after DHA supplementation (Table 4). Plasma TG level decreased by 18%, which did not reach statistical significance and was not as pronounced as the findings reported in other studies (25–30%) (Harris, 1997). The mild effect might be due to the low daily dose of n-3 fatty acids and original favorable lipid profiles in these postmenopausal vegetarians.

Lipid peroxides

The influence of n-3 fatty acids on lipid peroxidation in vivo depends on the balance of oxidative stress and induced antioxidative enzymes, but the net effect remains contradictory. This is probably due to the different doses of n-3 fatty acids used, different amounts of antioxidants supplied and different methods used to quantify the oxidative stress in previous studies. In this DHA oil, the amounts of α-tocopherol equivalent (α-TE) adjusted by numbers of double bonds were slightly lower than those in corn oil (Table 1), but its supplementation neither decreased α-tocopherol status nor increased urinary F2 isoprostane level (Table 4). F2 isoprostanes, a promising index of oxidative stress in vivo, are produced solely by free radical-induced peroxidation of AA (Morrow and Roberts, 1996). The unchanged AA status after DHA supplementation in this study was accompanied with an unchanged urinary F2-isoprostane level (Table 4), which represented no increase in AA peroxidation. Two research groups found a decrease in F2-isoprostane with higher doses of n-3 fatty acids than ours. One of these studies involved postmenopausal women supplemented with fish oil at a dose of 3.4 g n-3 fatty acids per day but significant differences of plasma F2-isoprostane were eliminated when the values were normalized to plasma AA concentrations (Higdon et al., 2000). Subjects in the other study were hypertensive type II diabetic patients (Mori et al., 2003). After having been supplemented with purified EPA or DHA at a dose of 4 g per day, their AA status and urinary F2-isoprostane was lower than the olive oil control group. Therefore, when the level of F2-isoprostane was used as a criterion, all the studies demonstrated no enhancement of in vivo oxidant stress after n-3 fatty acid supplementation. However, instead of F2-isoprostane, the peroxidation of EPA and DHA produced F3- and F4-isoprostanes, respectively (Nourooz-Zadeh et al., 1997, 1998). The influence of DHA supplementation on the production of F4-isoprostanes needs further evaluation. Some studies showing increased peroxidation and oxidative stress by n-3 fatty acid supplementation were suggested by an increase of LDL oxidation in vitro (Oostenbrug et al., 1994; Suzukawa et al., 1995). However, when similar assay was used to examine whether the observed unchanged in vivo oxidative stress status may be corroborated in vitro, results showed that the level of LDL-TBARS increased by DHA supplementation (Table 4). This would have been expected from the 1.75-fold enrichment of DHA in LDL (Table 4), as LDL-TBARS is produced by in vitro extensively oxidized LDL. The higher antioxidant status (Lu et al., 2000b; Manjari et al., 2001) of vegetarians might also have contributed to the resistance against oxidative stress from DHA.

Conclusion

This study shows that DHA supplementation at a dose of 2.14 g/day for 42 days decreases plasma cholesterol, but does not evidence to change the breast cancer risk marker measured as the ratio of urinary 2-OHE1 to 16α-OHE1, in postmenopausal vegetarian women. The in vivo antioxidant or oxidative stress markers measured as plasma α-tocopherol, and urinary F2-isoprostane are not deleteriously affected. However, this study had some limitations such as being a single-blind study with relative short duration, and small number of subjects. Therefore, longer period and larger number of subjects are needed to confirm these observations.

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Acknowledgements

This study was supported by a grant (Grant# DOH89-TD-1062) from Department of Health, Executive Yuan, Taiwan. We thank reviewers for the suggestion in statistical analysis and Professor Sieh-Hwa Lin from National Taiwan Normal University for assistance in statistical analysis.

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Correspondence to W H Wu.

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Guarantor: WH Wu.

Contributors: WHW designed the study, contributed to method development, data interpretation and preparation of the manuscript. SCL initiated the study, obtained a research grant for the study and was responsible for all stages of the study. TFW was responsible for biochemical and statistical analyses and taking care of the study subjects. HJJ and TAW were the gynecologists involved in subject recruitment, sample processing and advised on protocol design. All authors contributed to the writing of the manuscript.

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Wu, W., Lu, S., Wang, T. et al. Effects of docosahexaenoic acid supplementation on blood lipids, estrogen metabolism, and in vivo oxidative stress in postmenopausal vegetarian women. Eur J Clin Nutr 60, 386–392 (2006). https://doi.org/10.1038/sj.ejcn.1602328

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Keywords

  • DHA
  • algae oil
  • hydroxyestrone
  • blood lipids
  • oxidative stress
  • postmenopausal vegetarian women

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