Bixin uptake and antioxidative effect and role in immunoregulation in domestic cats.

Bixin, a carotenoid found in the seed of the Annatto plant, , is a potent antioxidant. Carotenoids are readily absorbed from the diet; therefore, the purpose of this study was to examine uptake of bixin by plasma, lipoproteins, and leukocytes after dietary supplementation in domestic cats and to assess effects on immune response. Female domestic short hair cats (3 yr old; 4.79 ± 0.13 kg BW) were fed a single dose of 0, 1, 5, or 10 mg bixin, and blood was taken at 0, 1, 2, 4 and 8 h after administration ( = 6/treatment) to determine acute absorption rate. Then, bixin was fed daily for 14 d to examine steady-state plasma concentrations and subcellular distribution. Following these preliminary experiments, cats ( = 8/treatment) were fed diets containing 0, 1, 5, or 10 mg bixin/d for 16 wk and blood was collected on wk 0, 6, 12, and 16 for analysis of leukocyte subpopulations, cell-mediated responsiveness, and inflammatory and oxidative biomarkers. Maximal uptake in plasma occurred 1 h after a single oral dose of bixin, with a maximal concentration of 0.119 µ and elimination half-life of 1.8 to 2.2 h. Daily feeding of bixin showed a steady-state plasma concentration of 0.110 µ at the greatest doses. Bixin was primarily associated with the high-density lipoprotein fraction of blood lipoproteins and was primarily distributed in mitochondrial fractions (58-59%) of but also in microsomal and nuclear fractions (37-44%). Leukocyte subpopulations in blood were variably affected by dietary bixin, with an increase ( < 0.05) in total T cells but a concurrent decrease ( < 0.05) in CD18+ and B cell subpopulations. However, plasma IgG increased ( < 0.05) in the 10-mg treatment group by wk 6. Lymphoproliferation was stimulated ( < 0.05) in the 5-mg bixin treatment group by wk 16, and delayed-type hypersensitivity response increased after nonspecific antigenic challenge. Conversely, when a specific challenge of vaccine was assessed on wk 12 and 16, responsiveness decreased ( < 0.05) in the 10-mg bixin treatment group. Bixin supplementation surprisingly caused an increase ( < 0.05) in α-acid glycoprotein but had no effect on natural killer cell activity, other subpopulations of leukocytes, or 8-oxo-2>-deoxyguanosine, a DNA damage biomarker. This experiment demonstrated dose-dependent uptake of bixin in plasma and blood lipoproteins and distribution in leukocyte subcellular components and an impacted immune response through cell-mediated and humoral actions.

Uptake and immunomodulatory role of bixin in dogs.

Carotenoids are readily absorbed from the diet and distributed in blood leukocyte subcellular organelles. Bixin, a potent bioactive found in the seed of the Annatto plant, , possesses antioxidant and anti-inflammatory properties. The purpose of this study was to determine the uptake of bixin by plasma, lipoproteins, and leukocytes in domestic dogs and to examine immunoprotective properties. To determine uptake kinetics, female Beagle dogs (2 yr; 9.1 ± 0.1 kg BW) were first fed a single dose by oral gavage of 0, 5, 10, 20, or 40 mg bixin, with blood collected at 0 to 16 h after administration ( = 6/treatment), and then fed daily with 0, 5, 10, 20, or 40 mg bixin/d, with blood collected at 0, 1, 2, 4, 6, 10, and 14 d. In a consecutive experiment, cell-mediated and humoral responses as well as oxidative biomarkers were measured following 16 wk of dietary supplementation with 0, 5, 10, or 20 mg bixin/d. Maximal absorption in plasma occurred by 0.5 h with an elimination half-life of 2.6 to 3.3 h after a single dose of bixin. Steady-state plasma concentrations were 0.053 µ after 14 d of 40 mg bixin/d. The majority of subcellular bixin was found in the leukocyte mitochondria and was associated with the high-density lipoprotein and low-density lipoprotein fractions of lipoproteins. Specific (vaccine) response increased ( < 0.05) but nonspecific mitogen response was unchanged after 12 wk of dietary bixin, as assessed by a delayed-type hypersensitivity assay. Both B cell plasma leukocyte subpopulations at 6 and 16 wk and IgG plasma concentration at 12 wk in the 10-mg treatment group increased ( < 0.05), although IgM production and other cell populations were unaffected. In addition, 8-oxo-2'-deoxyguanosine (8-OHdG), a DNA damage biomarker, was substantially reduced ( < 0.05) in all treatment groups by wk 16, and C-reactive protein (CRP) was suppressed at wk 12 ( < 0.05). Dietary supplementation with bixin showed no changes in lymphoproliferation in response to in vitro mitogenic challenge and had no effect in enhancing natural killer cell activity. In conclusion, bixin was readily absorbed in a dose-dependent manner in blood following oral administration and was then taken up by leukocytes, where it was primarily distributed to mitochondria but in other subcellular organelles as well. Bixin also appeared to stimulate immune response, as seen with cell-mediated responses, and exerted anti-inflammatory (reduced CRP) as well as antioxidative (reduced 8-OHdG) effects in dogs.

Astaxanthin modulates age-associated mitochondrial dysfunction in healthy dogs.

Young (2.97±0.01 yr; 8.16±0.15 kg BW) and geriatric (10.71±0.01 yr; 9.46±0.18 kg BW) healthy female Beagle dogs (n=14/age group) were fed 0 or 20 mg astaxanthin daily for 16 wk to examine modulation of mitochondrial function. Fasted blood was sampled on wk 0, 8, and 16. Mitochondria membrane permeability, ATP production, cytochrome c oxidase/reductase, and number were assessed in leukocytes whereas astaxanthin uptake, glutathione, superoxide dismutase, nitric oxide, 8-hydroxy-2'-deoxyguanosine, 8-isoprostane, and protein carbonyl were measured in plasma. Aging increased (P<0.05) complex III cytochrome c oxidoreductase but decreased (P<0.05) 8-hydroxy-2'-deoxyguanosine and protein carbonyl. Mitochondrial function improved in both young and geriatric dogs by increasing (P<0.05) ATP production, mitochondria mass, and cytochrome c oxidoreductase activity, especially in geriatric dogs compared with young dogs. Astaxanthin feeding also increased (P<0.05) the reduced glutathione to oxidized glutathione ratio in young dogs and decreased (P<0.05) nitric oxide in both young and geriatric dogs. Dietary astaxanthin improved mitochondrial function in blood leukocytes, most likely by alleviating oxidative damage to cellular DNA and protein.

The effects of soy milk and isoflavone supplements on cognitive performance in healthy, postmenopausal women.

RATIONALE: The decline in estrogen concentrations in women after menopause can contribute to health related changes including impairments in cognition, especially memory. Because of the health concerns related to hormone replacement therapy (HRT), alternative approaches to treat menopausal symptoms, such as nutritional supplements and/or diet containing isoflavones, are of interest. OBJECTIVES: This study investigated whether soy isoflavones (soy milk and supplement) could improve cognitive functioning in healthy, postmenopausal women. PARTICIPANTS, INTERVENTION AND DESIGN: A total of 79 postmenopausal women, 48-65 years of age, completed a double-blind, placebo-controlled trial in which they were randomly assigned to one of three experimental groups: cow's milk and a placebo supplement (control); soy milk and placebo supplement (soy milk, 72 mg isoflavones/day); or cow's milk and isoflavone supplement (isoflavone supplement, 70 mg isoflavones/day). MEASUREMENTS: Cognitive functioning was assessed using various cognitive tasks before the intervention (baseline) and after the intervention (test). RESULTS: In contrast to predictions, soy isoflavones did not improve selective attention (Stroop task), visual long-term memory (pattern recognition), short-term visuospatial memory (Benton Visual Retention Test), or visuo-spatial working memory (color match task). Also, the soy milk group showed a decline in verbal working memory (Digit Ordering Task) compared to the soy supplement and control groups. CONCLUSION: Soy isoflavones consumed as a food or supplement over a 16-week period did not improve or appreciably affect cognitive functioning in healthy, postmenopausal women.

Uptake of beta-carotene by ovarian and uterine tissues and effects on steroidogenesis during the estrous cycle in cats.

OBJECTIVES: To determine uptake of beta-carotene by ovarian and uterine tissues and influence of dietary beta-carotene on steroidogenesis and production of uterine protein during the estrous cycle in cats. ANIMALS: 56 female cats. PROCEDURE: Cats were fed diets containing 0, 0.4, 2, or 10 mg of beta-carotene daily for 8 weeks prior to detection of estrus. At time of observed estrus, all cats were manually induced to ovulate. Blood samples were obtained at estrus and every 2 days until day 14 after ovulation. On that day, cats underment laparotomy, and the ovaries and uterus were removed. Uterine contents were flushed, and luteal and endometrial tissues were obtained. RESULTS: Concentrations of beta-carotene in plasma and luteal and endometrial tissues increased in a dose-dependent manner. Concentrations of plasma progesterone were higher between days 6 and 10 after ovulation in cats fed diets containing beta-carotene and continued to increase through day 14 after ovulation in cats fed a diet containing 10 mg of beta-carotene. Plasma concentration of estradiol-17beta also was higher between days 0 and 4 after ovulation in cats fed diets containing beta-carotene. Cats fed a diet containing 10 mg of beta-carotene had the highest plasma estradiol concentration. Total uterine protein concentration was higher in cats fed beta-carotene, compared with values for cats fed an unsupplemented diet. CONCLUSION AND CLINICAL RELEVANCE: Cats readily absorb beta-carotene. Increased concentrations of progesterone, estradiol, and uterine protein may provide more optimal ovarian function or a better uterine environment for embryonic survival and development.

Dietary beta-carotene absorption by blood plasma and leukocytes in domestic cats.

Three experiments were conducted to study the uptake of oral beta-carotene by blood plasma and leukocytes in domestic cats. In Experiment 1, mature female Tabby cats (12 mo old) were given once orally 0, 10, 20 or 50 mg of beta-carotene and blood taken at 0, 12, 24, 30, 36, 42, 48 and 72 h after dosing. Concentrations of plasma beta-carotene increased in a dose-dependent manner. Peak concentrations were observed at 12-24 h and declined gradually thereafter. The half-life of plasma beta-carotene was 12-30 h. In Experiment 2, cats were dosed daily for six consecutive days with 0, 1, 2, 5 or 10 mg beta-carotene. Blood was sampled once daily at 12 h after each feeding. Daily dosing of cats with beta-carotene for 6 d resulted in a dose-dependent increase in circulating beta-carotene. Experiment 3 was designed to study the uptake of beta-carotene by blood leukocytes. Cats were fed 0, 5 or 10 mg of beta-carotene daily for 14 d. Blood leukocytes were obtained on d 7 and 14 to determine beta-carotene content in whole lymphocytes and in subcellular fractions. Blood lymphocytes took up large amounts of beta-carotene by d 7 of feeding. Furthermore, beta-carotene accumulated mainly in the mitochondria (40-52%), with lower amounts accumulating in the microsomes (20-35%), cytosol (15-34%), and nuclei (1.5-6%). Therefore, domestic cats readily absorb beta-carotene across the intestinal mucosa and transfer the beta-carotene into peripheral blood leukocytes and their subcellular organelles. beta-Carotene uptake kinetics show that some aspects of beta-carotene absorption and metabolism in cats are similar to those of humans.

Effects of dietary antioxidant supplementation on oxidative damage and resistance to oxidative damage during prolonged exercise in sled dogs.

OBJECTIVES: To determine effects of dietary antioxidant supplementation on plasma concentrations of antioxidants, exercise-induced oxidative damage, and resistance to oxidative damage during exercise in Alaskan sled dogs. ANIMALS: 62 Alaskan sled dogs. PROCEDURE: Dogs were matched for age, sex, and ability and assigned to 1 of 3 groups: sedentary and nonsupplemented (control [C]; n = 21), exercised and supplemented (S; 22), and exercised and nonsupplemented (N; 19). Dogs in group S were given 400 units of alpha-tocopherol acetate, 3 mg of beta-carotene, and 20 mg of lutein orally per day for 1 month, then dogs in groups S and N completed 3 days of exercise. Blood samples were collected before and after 1 and 3 days of exercise and after 3 days of rest. Plasma antioxidant concentrations were determined, and oxidative damage to DNA (plasma 7,8 dihydro-8-oxo-2'deoxyguanosine [8-oxodG] concentration) and membrane lipids (plasma hydroperoxide concentration) and resistance of plasma lipoproteins to oxidation were assessed. RESULTS: Supplementation increased plasma concentrations of alpha-tocopherol, beta-carotene, and lutein. Plasma concentration of alpha-tocopherol increased and concentration of lutein decreased in group S with exercise. Concentration of 8-oxodG decreased in group S but increased in group N during and after exercise. Lag time of in vitro oxidation of lipoprotein particles increased with exercise in group S only. CONCLUSIONS AND CLINICAL RELEVANCE: Dietary supplementation with antioxidants resulted in increased plasma concentrations of antioxidants. Moreover, supplementation decreased DNA oxidation and increased resistance of lipoprotein particles to in vitro oxidation. Antioxidant supplementation of sled dogs may attenuate exercise-induced oxidative damage.

Dietary beta-carotene stimulates cell-mediated and humoral immune response in dogs.

The role of beta-carotene on immune response in domestic dogs is not known. Female Beagle dogs were fed 0, 2, 20 or 50 mg beta-carotene/d; blood was sampled at wk 0, 1, 2, 4 and 8 for analysis of the following: lymphoproliferation, leukocyte subpopulations and concentrations of interleukin-2 (IL-2), immunoglobulin (Ig)G and IgM. Delayed-type hypersensitivity (DTH) response was assessed at wk 0, 3 and 7. beta-Carotene supplementation increased plasma beta-carotene concentrations in a dose-dependent manner. Compared with unsupplemented dogs, those fed 20 or 50 mg of beta-carotene had higher CD4+ cell numbers and CD4:CD8 ratio. However, there was no treatment difference in CD8+, CD21+ and major histocompatability complex (MHC) class II+ cells. Plasma IgG, but not IgM concentration was higher in dogs fed beta-carotene throughout the study period. The DTH response to phytohemagglutinin (PHA) and vaccine was heightened in beta-carotene-supplemented dogs. beta-Carotene feeding did not influence mitogen-induced lymphocyte proliferation or IL-2 production. Immune response was impaired in dogs classified as low beta-carotene absorbers compared with similar dogs fed the same amount of beta-carotene. Therefore, dietary beta-carotene heightened cell-mediated and humoral immune responses in dogs.

Yogurt consumption does not enhance immune function in healthy premenopausal women.

Fermented milk products may protect against breast cancer by stimulating immunologic activity. Twenty-five women [24.0 +/- 0.7 (SE) yr] were assigned randomly to two groups: control (n = 12) and yogurt treatment (n = 13). Controls refrained from yogurt products for three months, whereas the yogurt treatment group consumed two cups (454 g/day) of commercially produced yogurt for three consecutive months. Prior yogurt consumption did not exceed 4-6 cups/mo, and subjects consumed their usual diet during the study. Three-day diet records and fasting midluteal blood samples were obtained during subjects' first, second, and fourth menstrual cycles (baseline, Month 1, and Month 3, respectively). Macronutrient intakes differed between groups only for carbohydrate. Calcium intake increased for yogurt consumers during intervention. Lymphocyte proliferation induced by concanavalin A, phytohemagglutinin, and pokeweed mitogen, interleukin 2 production, and cytotoxic T lymphocyte-mediated cytotoxicity was assessed after baseline and Months 1 and 3 for both groups. No significant immune differences between the control and yogurt treatment group were observed for concanavalin A, phytohemagglutinin, pokeweed mitogen, interleukin-2, or cytotoxicity. In conclusion, three months of yogurt consumption did not enhance ex vivo cell-mediated immune function in young women.

Dietary beta-carotene is taken up by blood plasma and leukocytes in dogs.

beta-Carotene uptake by blood plasma and leukocytes was studied in mature beagle dogs. In expt. 1, dogs were fed once orally with 0, 50, 100 or 200 mg of beta-carotene and their blood was sampled at 0, 1. 5, 3, 6, 10, 18 and 24 h. Plasma beta-carotene concentrations increased dose-dependently to peak at 6 h postfeeding. Concentrations decreased rapidly thereafter, showing a half-life of 3 to 4 h. In expt. 2, dogs were given daily doses for seven consecutive days with 0, 12.5, 25, 50 or 100 mg beta-carotene. Plasma beta-carotene concentrations increased dose-dependently; concentrations after the last dose were two- to fourfold higher than after the first dose. In expt. 3, dogs were fed 0, 50 or 100 mg beta-carotene daily for 30 d. beta-Carotene was elevated in lymphocytes and neutrophils in supplemented dogs. Furthermore, beta-carotene was taken up by the cytosol, mitochondria, microsomes (lymphocytes and neutrophils) and nuclei (lymphocytes only), proving that dogs can absorb beta-carotene. beta-Carotene is taken up by subcellular organelles of blood lymphocytes and neutrophils and in the plasma and leukocytes beta-carotene may have physiological importance as it relates to immunity in dogs. Uptake kinetics indicated that dogs are not an appropriate animal model for studying beta-carotene absorption and metabolism in humans.

Beta-carotene uptake and changes in ovarian steroids and uterine proteins during the estrous cycle in the canine.

The uptake of beta-carotene by reproductive tissues and the effects of beta-carotene on reproductive function in the dog are unknown. We studied the uptake of beta-carotene by blood, corpus luteum, and uterine endometrium and the role of dietary beta-carotene in influencing ovarian steroid and uterine protein production during the estrous cycle in the dog. Mature female Beagle dogs (n = 56) were fed diets containing 0, 2, 20, or 50 mg of beta-carotene daily for approximately 6 wk before estrus detection. Blood was sampled at regular intervals from estrus through d 45 after ovulation (d 0 = ovulation), when laparotomy was performed. The ovaries were obtained for the isolation of corpus luteum. The uterus was flushed with phosphate-buffered saline and the endometrium obtained by scraping. Beta-carotene was not detectable in plasma, corpus luteum, or endometrium of unsupplemented dogs. However, beta-carotene and alpha-carotene in plasma, corpus luteum, and uterine endometrium increased in a dose-dependent manner. Alpha-carotene made up a high percentage of total carotenoids even though the alpha-carotene content in the dietary source was very low. Dogs fed 50 mg of beta-carotene had significantly higher concentrations of plasma progesterone between d 12 and 26 compared with unsupplemented dogs. Dietary beta-carotene did not influence plasma estradiol-17beta and total uterine proteins. Therefore, beta-carotene is absorbed into plasma, corpus luteum, and uterine endometrium of dogs. Furthermore, dietary beta-carotene increased plasma progesterone concentrations during the estrous cycle. It is possible that dietary beta-carotene may improve reproductive function in the canine.

Dietary lutein stimulates immune response in the canine.

The possible immuno-modulatory action of dietary lutein in dogs is not known. Female Beagle dogs (17-18-month old; 11.4+/-0.4kg body weight) were supplemented daily with 0, 5, 10 or 20mg lutein for 12 weeks. Delayed-type hypersensitivity (DTH) response to saline, phytohemagglutinin (PHA) and a polyvalent vaccine was assessed on Weeks 0, 6 and 12. Blood was sampled on Weeks 0, 2, 4, 8 and 12 to assess (1) lymphocyte proliferative response to PHA, concanavalin A (Con A), and pokeweed mitogen (PWM), (2) changes in peripheral blood mononuclear cell (PBMC) populations, (3) interleukin-2 (IL-2) production and (4) IgG and IgM production. After the completion of 12-week study, we continued to collect the blood weekly up to 17 weeks to evaluate the changes in immunoglobulin production upon first and second antigenic challenges on Weeks 13 and 15. Plasma lutein+zeaxanthin was undetectable in unsupplemented dogs but concentrations increased (P<0.05) rapidly on Week 2 in lutein-supplemented dogs. Thereafter, concentrations generally continued to increase in dose-dependent manner, albeit at a much slower rate. Dogs fed lutein had heightened DTH response to PHA and vaccine by Week 6. Dietary lutein increased (P<0.05) lymphocyte proliferative response to all three mitogens and increased the percentages of cells expressing CD5, CD4, CD8 and major histocompatibility complex class II (MHC II) molecules. The production of IgG increased (P<0.05) in lutein-fed dogs after the second antigenic challenge. Lutein did not influence the expression of CD21 lymphocyte marker, plasma IgM or IL-2 production. Therefore, dietary lutein stimulated both cell-mediated and humoral immune responses in the domestic canine.

Modulation of humoral and cell-mediated immune responses by dietary lutein in cats.

The immuno-modulatory role of dietary lutein in domestic cats is unknown. Female Tabby cats (10-month old; n=56) were supplemented daily for 12 weeks with 0, 1, 5 or 10mg lutein. Blood was collected on Weeks 0, 2, 4, 8 and 12 to assess the following: (1) mitogen-induced peripheral blood mononuclear cells (PBMCs) proliferation, (2) changes in PBMC subpopulations, (3) interleukin-2 (IL-2) production and (4) plasma immunoglobulin (Ig)G production. In addition, delayed-type hypersensitivity (DTH) response to concanavalin A (Con A) or a polyvalent vaccine was performed on Weeks 0, 6 and 12. Dietary lutein increased plasma lutein concentrations in a dose-dependent manner (p<0.001) and concentrations had not reached steady state after 12 weeks of feeding in cats given 5 or 10mg lutein. Concentrations of plasma retinol and alpha-tocopherol were not influenced by diet. The DTH response to vaccine but not to Con A increased (p<0.05) in a dose-dependent manner on Week 6. Compared to control, cats fed lutein also showed enhanced Con A- and pokeweed mitogen-stimulated PBMCs proliferation. Dietary lutein also increased the percentages of CD4+ and CD21+ lymphocytes on Week 12 but had no significant effect on pan T, CD8 and MHC class II markers. Plasma IgG was higher (p<0.05) in cats fed 10mg lutein on Weeks 8 and 12. These results support the immuno-modulatory action of lutein in domestic cats.

A standardized gating technique for the generation of flow cytometry data for normal canine and normal feline blood lymphocytes.

Flow cytometry is becoming a commonly used technique to characterize a variety of cells. It provides a powerful application to rapidly determine the relative percentages of T-lymphocyte subsets and B-lymphocytes. The effectiveness of its application, however, is dependent on standardization, especially in a clinical setting. Application of flow cytometry to veterinary diagnostics has been limited by the unavailability of reagents and by the unstandardized characterization of normal values using antibodies not commercially available, but typically provided through the generosity of other researchers. This paper presents a standardized gating protocol, and average values and ranges observed for normal canine and feline blood lymphocytes using commercially available antibodies to cell surface markers for CD5, CD3, CD4, CD8, MHC II, and B lymphocytes. The averages for these markers on gated lymphocytes were as follows: Canine CD5 83.3%, Canine CD4 45.0%, Canine CD8 28.8%, Canine MHC II 98.0%, Canine B Cell 12.9%, Canine CD4/CD8 ratio 1.87, Feline T lymphocytes 77.3%, Feline CD4 44.5%, Feline CD8 25.7%, Feline B Cell 24.1%, Feline CD4/CD8 Ratio 1.75. Normal values were also established for a mixed breed group of dogs, and old versus young dogs. This information will provide researchers and clinicians with a standardized protocol for gating, which establishes a basis for comparison between techniques, and a measure of phenotypic percentages for flow cytometry in normal dogs and cats based on this standardization and commercially available antibodies.

A comparison of the anticancer activities of dietary beta-carotene, canthaxanthin and astaxanthin in mice in vivo.

The anticancer activities of beta-carotene, astaxanthin and canthaxanthin against the growth of mammary tumors were studied in female eight-wk-old BALB/c mice. The mice were fed a synthetic diet containing 0, 0.1 or 0.4% beta-carotene, astaxanthin or canthaxanthin. After 3 weeks, all mice were inoculated with 1 x 10(6) WAZ-2T tumor cells into the mammary fat pad. All animals were killed on 45 d after inoculation with the tumor cells. No carotenoids were detectable in the plasma or tumor tissues of unsupplemented mice. Concentrations of plasma astaxanthin (20 to 28 mumol/L) were greater (P < 0.05) than that of beta-carotene (0.1 to 0.2 mumol/L) and canthaxanthin (3 to 6 mmol/L). However, in tumor tissues, the concentration of canthaxanthin (4.9 to 6.0 nmol/g) was higher than that of beta-carotene (0.2 to 0.5 nmol/g) and astaxanthin (1.2 to 2.7 nmol/g). In general, all three carotenoids decreased mammary tumor volume. Mammary tumor growth inhibition by astaxanthin was dose-dependent and was higher than that of canthaxanthin and beta-carotene. Mice fed 0.4% beta-carotene or canthaxanthin did not show further increases in tumor growth inhibition compared to those fed 0.1% of each carotenoid. Lipid peroxidation activity in tumors was lower (P < 0.05) in mice fed 0.4% astaxanthin, but not in those fed beta-carotene and canthaxanthin. Therefore, beta-carotene, canthaxanthin and especially astaxanthin inhibit the growth of mammary tumors in mice; their anti-tumor activity is also influenced by the supplemental dose.

Dietary lutein but not astaxanthin or beta-carotene increases pim-1 gene expression in murine lymphocytes.

This study investigates the effect of dietary carotenoids on pim-1 gene expression in mouse splenocytes. Female BALB/c mice were fed 0%, 0.02%, or 0.4% astaxanthin, beta-carotene, and lutein for two weeks. Plasma and liver were obtained for the analysis of carotenoids. Splenocytes were isolated and cultured in the presence of concanavalin A, and the level of pim-1 mRNA was determined by Northern blot analysis. None of the carotenoids were detectable in the plasma and liver of unsupplemented mice. In plasma the concentration of astaxanthin (4.9-54.7 mumol/l) was dramatically higher than that of lutein (1.4-2.0 mumol/l) and beta-carotene (0.1-0.7 mumol/l). Carotenoid uptake by the spleen but not the liver reflected that observed in plasma. In mice fed 0.4% of each carotenoid, the absolute concentration of the carotenoid in the liver was highest for astaxanthin (24 nmol/g) followed by beta-carotene (7.5 nmol/g) and lutein (1.58 nmol/g). Mice fed lutein showed a dose-related increase in pim-1 mRNA expression. The steady-state level of pim-1 mRNA in mice fed 0.4% lutein was sixfold higher than in mice fed 0.02% lutein. In contrast, dietary astaxanthin and beta-carotene did not affect pim-1 expression. Therefore, an increase in pim-1 mRNA was observed in splenocytes stimulated with concanavalin A in lutein-fed mice. This appears to be a unique effect of lutein and may be associated with its antitumor activity observed in vivo.

Dietary beta-carotene and astaxanthin but not canthaxanthin stimulate splenocyte function in mice.

The in vivo modulatory effect of beta-carotene, astaxanthin and canthaxanthin on lymphocyte function was investigated. Female BALB/c mice (8 wk old) were fed a basal diet containing 0, 0.1% or 0.4% beta-carotene, astaxanthin or canthaxanthin for 0, 2 or 4 wk (n = 8/diet/period). Splenic lymphocytes were isolated and mitogen-stimulated proliferation, IL-2 production and lymphocyte cytotoxicity were assessed. Body weight and feed intake were not different among dietary treatments. Plasma carotenoids were undetectable in unsupplemented mice but concentrations of the respective carotenoids were elevated in mice fed 0.1 or 0.4% beta-carotene (0.22 and 0.39 mumol/L), astaxanthin (16.4 and 50.2 mumol/L) and canthaxanthin (5.00 and 7.02 mumol/L) respectively. Mice fed both dietary levels of beta-carotene and astaxanthin had enhanced phytohemagglutinin-induced lymphoblastogenesis compared to unsupplemented mice (P < 0.03). No treatment difference was detected with concanavalin A- or lipopolysaccharide-induced lympho-proliferation nor with IL-2 production (P < 0.05). Astaxanthin (0.1%) also enhanced lymphocyte cytotoxic activity (P < 0.08). In contrast, canthaxanthin did not significantly influence any of the lymphocyte functions measured. Results indicate that beta-carotene and astaxanthin but not canthaxanthin exert enhanced splenic lymphocyte function in mice.

Dietary lutein from marigold extract inhibits mammary tumor development in BALB/c mice.

High levels of dietary lutein can inhibit mammary tumor growth in mice. However, the antitumor effect of low levels of dietary lutein on mammary tumors is unavailable. Female BALB/c mice and the WAZ-2T (-SA) mammary tumor cell line were used in two experiments. A preliminary tumor cell dose titration study (Experiment 1) was designed to determine the inoculation dose to produce approximately 65% tumor incidence. Mice (n = 10/dose) were inoculated with 0 to 1 x 10(6) tumor cells in the right inguinal mammary fat pad. A tumor cell load of 2.5 x 10(3) cells/inoculation produced approximately 65% tumor incidence. This dose was used in a subsequent study (Experiment 2) of the efficacy of dietary lutein against mammary tumor development. Mice (n = 20/treatment) were fed a semisynthetic diet containing 0, 0.002, 0.02, 0.2 or 0.4% lutein from marigold extract. After 14 d, all mice were inoculated with 2.5 x 10(3) tumor cells, and tumor growth was measured daily for 70 d at which time blood, liver, spleen and tumors were obtained. Lutein + zeaxanthin uptake increased dose-dependently (P < 0.05) with dietary lutein levels from 0 to 0.02% (spleen) or 0.2% (plasma, liver and tumor). Low levels (0.002 and 0.02%) of dietary lutein lowered (P < 0.05) mammary tumor incidence, tumor growth and lipid peroxidation, and increased tumor latency, whereas higher dietary levels (0.2 or 0.4%) were less effective. Therefore, very low amounts of dietary lutein (0.002%) can efficiently decrease mammary tumor development and growth in mice.

Dietary lutein absorption from marigold extract is rapid in BALB/c mice.

Even though lutein can stimulate immunity and decrease cancer growth, no systematic studies are available on the uptake of lutein in mice. We studied the uptake of lutein in 8-wk-old female BALB/c mice fed a diet containing 0, 0.05, 0.1, 0.2 or 0.4% lutein. Mice were killed on d 0, 3, 7, 14, 21 and 28 (n = 6/period), and blood, spleen and liver were collected. Food intake and body, liver and spleen weights did not differ among treatment groups. Lutein + zeaxanthin were not detectable in the plasma, liver and spleen of unsupplemented mice. Mice fed lutein showed very rapid lutein + zeaxanthin absorption. On d 3, concentrations of plasma lutein + zeaxanthin had rapidly increased (P < 0.05) in lutein-fed mice and no further increases were observed. Plasma lutein + zeaxanthin concentrations did not differ among lutein-fed mice by d 7 (2.58 +/- 0.2 micromol/L). Even though maximal uptake of plasma lutein + zeaxanthin was observed by d 3, uptake of lutein + zeaxanthin by the liver and especially by the spleen generally continued to increase (P < 0.05) through d 28 to reach concentrations of 0.11 +/- 0.001 (spleen) and 0.71 +/- 0. 0002 (liver) nmol/g. Therefore, dietary lutein is readily absorbed into the plasma and taken up by liver and spleen of mice. Plasma lutein + zeaxanthin concentrations were higher than in human studies; however, mice were fed lutein at a level several hundredfold greater than in humans. The liver is a major storage organ for lutein + zeaxanthin in mice. Uptake of lutein + zeaxanthin by the spleen suggests a role for lutein in modulating immunity.

Zinc oxide and amino acids as sources of dietary zinc for calves: effects on uptake and immunity.

Calf starter diets were formulated to contain 60 ppm of Zn, 150 or 300 ppm of Zn in the form of Zn-Met and Zn-Lys, or 300 ppm of Zn in the form of ZnO to compare relative bioavailability and effects on immunity. Holstein heifer calves were weaned at wk 5 and fed experimental starter diets from wk 6 to 12. Feed intake, body weight, Zn concentrations in liver and serum fractions, and mineral concentrations in serum were measured to determine the effects of treatment. In addition, peripheral blood lymphocyte blastogenesis, interleukin-2 production, cytotoxic activity, and the ability of blood neutrophils to phagocytose and kill bacteria were assessed at wk 0, 2, 4, and 6 of the trial. Feed intakes and body weight gains were similar among calves. Concentrations of Zn in serum were elevated in calves fed 300 ppm of Zn as Zn-Met and Zn-Lys but not in calves fed ZnO. Concentrations of Zn in liver were significantly elevated by 300 ppm of Zn in the form of Zn-Met and Zn-Lys (360 micrograms/g) but not by the other Zn treatments or by the control (245 micrograms/g). No treatment had an effect on the concentrations of Lys and Met in serum; however, concentrations of Lys did decrease in serum as the age of the calves increased. There was no significant treatment effect on mitogen-induced lymphocyte blastogenesis, interleukin-2 production, lymphocyte cytotoxicity, or phagocytic and intracellular killing ability of blood neutrophils. These data indicated greater absorption and retention of Zn when administered in the form of Zn-Met and Zn-Lys than that when ZnO was administered to young calves. However, there was no advantage to the immune function of extra dietary Zn.