点击显示 收起
1 From the Department of Nutrition and Food Science and the Biometrics Program, the Department of Animal and Avian Sciences, University of Maryland, College Park, and the US Department of Agriculture, Agricultural Research Service, Beltsville Human Nutrition Research Center, Phytonutrients Laboratory, Beltsville, MD.
2 Supported in part by Maryland Agricultural Experiment Station grant NFSC-96-39 and the US Department of Agriculture, Agricultural Research Service. Hoffman-La Roche (Nutley, NJ) donated the ß-carotene supplements and the placebo. 3 Address reprint requests to PB Moser-Veillon, Department of Nutrition and Food Science, University of Maryland, College Park, MD 20742. E-mail: pv6{at}umail.umd.edu.
ABSTRACT
Background: Information is lacking regarding the effects of ß-carotene supplementation, early lactation, or both on circulating carotenoid concentrations and T lymphocyte proliferation.
Objectives: This study investigated the effects of short-term ß-carotene supplementation (30 mg/d for 28 d) during early lactation (days 4–32 postpartum) on circulating carotenoid concentrations and on the T lymphocyte proliferative response to phytohemagglutinin.
Design: Subjects aged 19–39 y were paired [lactating (4 d postpartum) and nonlactating (never pregnant, healthy women)] and randomly assigned to receive either ß-carotene or a placebo. During the study, subjects provided eight 24-h food records for analysis with the NUTRITIONIST IV and US Department of Agriculture carotenoid databases. Nonfasting blood samples were collected at baseline and at 28 d. Plasma analysis included quantification of -carotene, ß-carotene, lutein, lycopene, retinol, and -tocopherol, complete differential blood cell counts, and lymphocyte proliferative activity.
Results: ß-Carotene supplementation increased ß-carotene (P < 0.001) and -carotene (P < 0.05) concentrations but did not affect lycopene concentrations significantly. Supplemented women showed significant decreases in plasma lutein (P < 0.03), as did lactating subjects (P < 0.02). Neither lactation nor ß-carotene supplementation affected the T lymphocyte proliferative response to phytohemagglutinin.
Conclusions: Our results suggest that ß-carotene supplementation as well as some events related to parturition, initiation of lactation, or both alter circulating concentrations of lutein. ß-Carotene supplementation does not enhance T lymphocyte immune competence in healthy women.
Key Words: ß-Carotene • carotenoids • lactation • lutein • T lymphocyte proliferation
INTRODUCTION
The effect of ß-carotene supplementation on the metabolism and plasma concentrations of other major carotenoids such as -carotene, lutein, and lycopene remains equivocal. This may be due in part to the complex relations between factors known to affect the absorption and metabolism of carotenoids. These factors include carotenoid intake, vitamin A status, and individual variability (1–4). Evidence of fluctuations in plasma carotenoid concentration throughout the menstrual cycle (5) suggests that carotenoid concentrations may also respond to altered reproductive physiology such as pregnancy and lactation.
Clinical studies examining the effect of ß-carotene supplementation on immune function also failed to provide conclusive results. Although many studies have reported an enhancement in markers of immune activation such as lymphocyte proliferation or cytokine production after ß-carotene supplementation (6–9), several more recent reports have not (10–12). Growing evidence suggests that the lactogenic hormone prolactin is also involved in the regulation of immune response in humans (13–15): B and T lymphocytes express high-affinity receptors for prolactin and produce a prolactin-like substance with similar immunomodulatory properties. Lactation itself is associated with local immunoactivation of the mammary gland, and immune cells present in breast milk exhibit activation markers (16). Therefore, the experiment was designed to explore possible interactions between short-term ß-carotene supplementation and lactation on mitogenic DNA synthesis in immune-related peripheral blood T lymphocytes as well as to determine the effects of ß-carotene supplementation on concentrations of other major carotenoids in the plasma of lactating and nonlactating women.
SUBJECTS AND METHODS
Subjects
This study was approved by the University of Maryland Institutional Review Board as conforming to the University and Public Health Service Policy in protecting the rights of study participants. Written, informed consent was obtained from each subject before participation in the study. Seventeen postpartum, lactating women who had lactated previously and 17 never-pregnant women aged 19–39 y were recruited as study participants. Because of technical difficulties with blood collection and storage, results are reported for only 16 postpartum women. Subjects were nonsmokers, did not use oral contraceptives, and did not take ß-carotene supplements for 3 mo before the start of the study. Subjects entered the study as pairs: one lactating and one nonlactating woman. Women were paired according to the availability of the nonlactating member on day 4 postpartum of the lactating member. Each pair was randomly assigned to receive either ß-carotene supplements (30 mg/d) or placebo and began the study on day 4 postpartum of the lactating member. Overall, 8 lactating and 8 nonlactating women received a placebo, whereas 9 lactating and 9 nonlactating women received ß-carotene supplements (Hoffmann-La Roche, Nutley, NJ) for 28 d at breakfast with 250 mL whole milk.
Dietary intake
Each subject provided 24-h food records on days 0, 3, 6, 9, 12, 15, 21, and 27. The food records were analyzed with NUTRITIONIST IV software (N-Squared Computing, Salem, OR) to determine average intakes of energy and vitamin A and the percentages of energy from fat, carbohydrates, and protein in the diet. The food records were also analyzed with the US Department of Agriculture's carotenoid database for fruit, vegetables, and multicomponent foods (2) to determine intakes of -carotene, ß-carotene, lycopene, and lutein.
Carotenoid analysis
Nonfasting blood samples (15 mL each) were collected by venipuncture on days 0 and 28 and were used for biochemical and immunologic assays. Blood for HPLC determination of carotenoids and hematologic indexes was collected into vacuum tubes coated with EDTA. Plasma was obtained by refrigerated centrifugation (680 x g for 12 min at 4°C) and stored at -80°C until analyzed. Measured amounts of internal standard (ß-apo-8-carotenal in ethanol containing 1 g butylated hydroxytoluene/L) were added to each plasma sample. Samples were then extracted 3 times with hexane; combined hexane extracts were filtered and dried under nitrogen. Samples were reconstituted with 500 µL of mobile phase (65% acetonitrile, 25% methylene chloride, and 10% methanol) and analyzed with a 1050 series HPLC instrument (Hewlett-Packard, Palo Alto, CA) equipped with a diode array detector (17).
The accuracy of calibration curves was validated by analyzing standard reference materials (SRM 968B, human serum; National Institute of Standards and Technology, Gaithersburg, MD). Plasma concentrations of individual carotenoids (-carotene, ß-carotene, lutein, and lycopene), retinol, and -tocopherol were corrected according to recovery of the internal standard. Compliance with the supplementation protocol was assessed by HPLC analysis of plasma ß-carotene concentrations. ß-Carotene and placebo capsules were assayed to confirm their carotenoid contents.
Mitogenic T lymphocyte proliferation
The response of T lymphocytes ex vivo to phytohemagglutinin was assayed by using the whole-blood culture method as described by Kramer and Burri (18). Briefly, fresh whole blood was collected into vacuum tubes coated with heparin and then diluted 1:4 with tissue culture medium containing 2.0 µmol L-glutamine/L with 100 x 103 U penicillin/L and 100 mg streptomycin/L (Sigma Chemical Co, St Louis). This dilution was used to prepare an 8-point dose-response curve to phytohemagglutinin (0, 1.25, 2.5, 5, 10, 20, 40, and 80 mg/L). Cultures were incubated for 72 h. [3H]Thymidine (37 kBq, or 1 µCi) (Dupont NEN Products, Boston) was added 24 h before cell cultures were harvested (Skatron, Inc, Sterling, VA) onto fiberglass filters. The [3H]thymidine-labeled DNA on individual filtermate discs with scintillant (Ready Safe; Beckman, Palo Alto, CA) was counted in a Beckman LS 3801 scintillation counter by using a single-label program with the activity reported as dpm/culture. The median value of each triplicate culture is reported as log10 dpm. Proliferation activity was also evaluated per lymphocyte (log10 dpm/lymphocyte).
Statistical analysis
Statistical analysis was carried out with SAS software (version 6.11; SAS Institute Inc, Cary, NC) and all analyses were done by using the mixed-model procedure. Most data were organized within a 2 x 2 factorial model (lactating compared with nonlactating and ß-carotene compared with placebo). Carotenoid concentrations and leukocyte count data were analyzed as a completely randomized split plot in time. Comparisons among least-squares means were tested at the 5% level of significance.
RESULTS
Subjects were primarily white, except for 6 Asian or Indian, 2 Hispanic, and 2 African American women. All subjects lived in the metropolitan Washington, DC, area. Lactating women had a higher (P < 0.002) mean body mass index (in kg/m2) and were older (P < 0.01) than non lactating women (Table 1). Lactating women also had significantly higher dietary intakes of energy (P < 0.002), percentages of energy from fat (P < 0.01; data not shown), vitamin A (P < 0.001), and ß-carotene (P < 0.04). There were no significant differences in intakes of -tocopherol, -carotene, lycopene, or lutein between lactating and nonlactating women (Table 2). HPLC analysis of capsule extracts as prepared by 2 different technicians confirmed the dose to be between 31 and 35 mg/capsule. Results of capsule analysis revealed that the supplements were between 82% and 91% ß-carotene.
View this table:
TABLE 1.. Characteristics of subjects at baseline1
View this table:
TABLE 2.. Mean daily intake from eight 24-h food records of selected nutrients during the 28-d study1
Subject compliance regarding supplement administration was confirmed by plasma ß-carotene concentrations. At the end of the study period, these concentrations had risen 800% in the lactating women and 900% in the nonlactating women (P < 0.001). Plasma ß-carotene did not change significantly from baseline in the unsupplemented women (Figure 1).
FIGURE 1. . Mean (±SE) plasma ß-carotene concentrations in lactating and nonlactating women after 28 d of supplementation with ß-carotene (30 mg/d) or placebo. *Significantly different from baseline, P < 0.001. Note that SEs for supplemented women at baseline were too small to depict on the figure.
ß-Carotene supplementation, regardless of physiologic status, resulted in a significant increase (P < 0.05) in plasma -carotene concentrations (Figure 2). Supplementation and lactation both resulted in significant decreases in plasma lutein (P < 0.03; Figure 3). These decreases were not reflected in lutein intake during the study, ie, there were no significant changes in lutein intake over time in any of the groups. ß-Carotene supplementation did not significantly affect the concentrations of lycopene, -tocopherol, or retinol (data not shown).
FIGURE 2. . Mean (±SE) plasma -carotene concentrations in lactating and nonlactating women after 28 d of supplementation with ß-carotene (30 mg/d) or placebo. *Significantly different from baseline, P < 0.05.
FIGURE 3. . Mean (±SE) plasma lutein concentrations in lactating and nonlactating women after 28 d of ß-carotene supplementation (30 mg/d) or placebo. *Significantly different from baseline, P < 0.03. There was no significant interaction between lactation status and ß-carotene supplementation.
ß-Carotene supplementation had no significant effect on absolute numbers or percentages of leukocytes in either group. ß-Carotene supplementation did not significantly affect the mitogenic proliferative responsiveness of T lymphocytes per volume of blood in whole-blood microcultures to phytohemagglutinin in lactating or nonlactating women (Figure 4). This was also true when the activity was evaluated per peripheral blood lymphocyte (data not presented).
FIGURE 4. . Mean (±SE) T lymphocyte proliferation in lactating and nonlactating women after 28 d of supplementation with ß-carotene (30 mg/d) or placebo. There were no significant differences from baseline in any of the groups.
At baseline, the lactating women had greater numbers of total leukocytes (P < 0.004) and polymorphonuclear leukocytes than did the nonlactating women (P < 0.005). Treatment with ß-carotene had no significant effect on the absolute numbers or percentages of different types of leukocytes.
DISCUSSION
Energy and vitamin A intakes were consistent with previously reported intakes in lactating (19) and nonlactating premenopausal women (20). Carotenoid intakes of the postpartum women were consistent with other reports of lactating women (21, 22). Among the nonlactating women, lutein intakes were within reported values; mean (±SD) intakes of ß-carotene (3461 ± 2510 µg/d) and lycopene (4074 ± 4938 µg/d) appeared to be slightly higher than those reported for other premenopausal women. Reported mean values ranged from 2060 to 2653 µg/d for ß-carotene and from 867 to 3056 µg/d for lycopene (2, 5, 23, 24). The mean -carotene intake of nonlactating women in the present study (651 ± 591 µg/d) appeared to be much higher than other reported values (range: 105–573 µg/d; 2, 5, 23, 24), although the variability was also high. Although we did not perform a meta-analysis, it is unlikely that the differences between studies are significant because of the high degree of intraindividual variability.
Plasma concentrations of retinol and -tocopherol were consistent with values reported for healthy, well-nourished, lactating (21, 22) and nonlactating (3, 25) women. Among nonlactating women, baseline plasma concentrations of ß-carotene were within reported ranges. In the present study, -carotene concentrations were higher (0.19 ± 0.18 µmol/L) and lycopene concentrations were lower (0.46 ± 0.23 µmol/L) than reported means of 0.02–0.11 µmol/L and of 0.58–0.76 µmol/L, respectively (2, 3, 5, 26, 27) in nonlactating women. Among lactating women, baseline plasma -carotene, lycopene, and lutein concentrations were comparable with previously reported values, but plasma ß-carotene concentrations appeared to be lower than in previous studies (21, 22).
Supplementation resulted in a 9-fold increase in plasma ß-carotene concentrations in nonlactating subjects and an 8-fold increase in lactating subjects. This response to supplemental ß-carotene has been reported in many study populations. Specifically, Canfield et al (22) observed a 7.4-fold increase in plasma ß-carotene in lactating women supplemented with identical amounts of ß-carotene for the same duration (28 d). The significant increase in plasma -carotene concentrations observed after supplementation was reported previously (28) and is assumed to be, at least in part, due to contamination of the supplements. Plasma lycopene concentrations did not change significantly after ß-carotene supplementation in our study (data not shown) and were comparable with values from 2 other studies (21, 22).
Plasma lutein decreased significantly in response to ß-carotene supplementation. This phenomenon was reported by several researchers and probably reflects decreased lutein absorption. Plasma lutein decreased in lactating women regardless of supplementation status. The changes in plasma lutein appeared to be independent of lutein intake, at least during the study period, because lutein intakes did not change significantly over time in any of the groups. It is possible that lutein intakes were lower during the study than during the weeks before the study; however, we do not have those data for comparison. An alternative explanation for the decrease might be that lipoprotein concentrations were declining. Although we did not measure lipoproteins, decreasing concentrations were not likely in the nonlactating women because of the short duration of the study and because the subjects were premenopausal. Furthermore, although a decrease in plasma lipoprotein concentrations would account for a decrease in plasma lutein, it would also be expected to result in decreases in other carotenoids; however, this was not observed. Thus, we conclude that postpartum normalization of lipoprotein concentrations did not explain the changes in plasma lutein. One intriguing observation was the significant decrease in plasma lutein observed in both supplemented and unsupplemented lactating women. This result suggests that some event or set of events associated with parturition, initiation of lactation, or both may affect lutein absorption, metabolism, or both. These events might include changes in carotenoid or other nutrient intakes, postpartum normalization of lipoprotein concentrations, or changes in adiposity. It is difficult to speculate about the nature of these events on the basis of the limited data we collected. These limitations include the fact that the food records were self-reported estimates of intake and the US Department of Agriculture database of the carotenoid content of commonly consumed foods, although an important advance, provides relatively crude estimates of carotenoid intake.
Leukocyte counts were well within the hematologic reference ranges (25th–75th percentiles) reported for women of various racial and ethnic backgrounds (29). The significant shifts observed in the numbers of total leukocytes and polymorphonuclear leukocytes in lactating women were most likely due to postpartum normalization (30). Neither ß-carotene supplementation nor lactation had a significant effect on mitogen-induced proliferative activity of peripheral blood lymphocytes. Reports of the effect of ß-carotene supplementation on lymphocyte proliferation have been conflicting, partly because of differences in the duration of supplementation and the use of different assays. The most common method of cell culture for measuring lymphocyte proliferation is the separated peripheral blood mononuclear cell (PBMNC) technique (31), which involves the addition of heterologous plasma to the isolated PBMNC fraction. In contrast, the whole-blood method used in this study uses cells in autologous plasma and thus is probably a better indicator of the in vivo immune response.
Notwithstanding methodologic differences, our results agree with those reported by Daudu et al (11) and Ringer et al (10), who found no significant effect of ß-carotene supplementation on lymphocyte function or lymphocyte subsets with use of a traditional separated cell culture. On the other hand, Moriguchi et al (8) found an increase in peripheral blood lymphocyte proliferation after supplementing young men with 30 mg ß-carotene/d for 1 mo. The separated peripheral blood lymphocyte technique used by Moriguchi et al is a variation of the traditional separated PBMNC method taken one step further by isolating peripheral blood lymphocytes by removing plastic-adherent monocytes. It is possible that the resulting increase in the percentage of lymphocytes amplified proliferative responsiveness.
Beyond methodologic differences, these conflicting results may represent differences in oxidative stress or antioxidant status. Van Poppel et al (9) found elevated T lymphocyte proliferation in male smokers supplemented with ß-carotene; smokers often exhibit marginal to poor antioxidant status, perhaps because of an increased oxidant burden (32). Kramer and Burri (18) found that mitogenic T cell proliferative response decreased in young women after they consumed a low-carotenoid diet for 60 d; supplementation with mixed carotenoids reversed this decrease. Supplemental ß-carotene appears to enhance mitogenic T lymphocyte proliferation in those people known or suspected to have suboptimal circulating carotenoid or other antioxidant concentrations. Cross et al (12) found, however, that the same mixed carotenoid supplement used by Kramer and Burri had no significant effect on mitogenic T cell proliferation in a free-living group of elderly women with normal plasma carotenoid profiles at baseline. Our subjects were nonsmoking, healthy young women who entered the study with normal intakes and plasma concentrations of key antioxidants: vitamins C and E, ß-carotene, and other carotenoids. These data support the finding that additional ß-carotene has no effect on T lymphocyte proliferation in people known to have normal plasma carotenoid concentrations.
ß-Carotene supplementation and lactation appear to affect the plasma concentrations of lutein. The decrease observed in supplemented subjects may reflect impaired lutein absorption after ß-carotene supplementation. The significant decrease in plasma lutein in lactating women is particularly intriguing and further investigation is needed to elucidate the causes of this decrease. Our data indicate that this postpartum normalization of plasma lutein concentration happens during the first month after delivery.
The proliferative response of blood lymphocytes to phytohemagglutinin was not affected significantly by ß-carotene supplementation or lactation. These data support recent studies suggesting that the effects of ß-carotene supplementation on mitogenic T lymphocyte proliferation are found only in subjects with suboptimal antioxidant status.
ACKNOWLEDGMENTS
We thank E Wiley and M Howard, Beltsville Human Nutrition Research Center, for excellent technical assistance with the carotenoid and lymphocyte proliferation analyses, respectively.
REFERENCES