Suppression of tumor growth and enhancement of immune status with high levels of dietary vitamin B6 in BALB/c mice.

Effects of dietary vitamin B6 at levels ranging from deficiency to megadoses on the development of herpes simplex virus type 2-transformed (H238) cell-induced tumors and on in vitro responses relating to cell-mediated immunity were examined. Male BALB/cByJ mice (n = 260), 5 weeks of age, were fed 20% casein diets containing pyridoxine (PN) at 0.2, 1.2 for the control diet, 7.7, or 74.3 mg/kg diet for 4-11 weeks. After 4 weeks of dietary treatment, 120 of the mice received an injection of H238 cells; mice without H238 injection served as controls. At 4, 8, and 11 weeks, animals from each group were euthanized and blood and spleen samples obtained. Mice fed 0.2 mg PN developed mild deficiency symptoms and gained significantly less weight than those fed 1.2-, 7.7-, and 74.3-mg PN diets. Thirteen to 16 days after tumor cell injection, primary tumor incidence was lowest in mice fed 74.3 mg PN; later, incidence among groups was similar. Mice fed 1.2 mg PN had the largest primary tumor volume, the highest incidence of lung metastases, and the greatest number of metastatic nodules per animal at 7 weeks post injection. Overall, lower tumor volumes were found in animals fed 7.7 and 74.3 mg PN (14 and 32% less than the tumor volume for those fed 1.2 mg PN, respectively); mice fed 0.2 mg PN had the lowest tumor volume. Blood and spleen lymphoproliferative response to stimulation by phytohemagglutinin or concanavalin A generally tended to be higher in mice fed 7.7 and 74.3 mg PN as compared to that in animals fed either 0.2 or 1.2 mg PN. However, decreased mitogen-stimulated responsiveness was observed in all animals with progressive tumor growth. Tumor growth also resulted in splenomegaly and increased thymic atrophy. Significant negative relationships between tumor volume and tumor pyridoxal 5-phosphate (PLP) concentrations were observed for 1.2-, 7.7-, and 74.3-mg PN diet groups. These data suggest that high dietary intake of vitamin B6 may have suppressed tumor development by either immune enhancement or PLP growth regulation of this tumor.

Physiological free fatty acid concentrations do not increase free estradiol in plasma.

Consumption of dietary fat has been linked to the high incidence of breast cancer. The in vitro effects of palmitate, oleate, linoleate, and eicosapentaenoate at supraphysiological concentrations [1.8-4.8 mM; endogenous (0.8) plus added (1-4)] on the binding of 17 beta-estradiol (E2) to sex hormone-binding globulin and albumin were evaluated in plasma, using centrifugal ultrafiltration. The E2-sex hormone-binding globulin complex was significantly dissociated by oleate, linoleate, eicosapentaenoate, and palmitate at 1.8 to 2.8-mM concentrations. The same FFA caused a significant increase in E2 binding to albumin at similar concentrations. However, only the unsaturated fatty acids at 3.8 to 4.8-mM concentrations (i.e. oleate, linoleate, and eicosapentaenoate) significantly increased the nonprotein-bound free E2 fraction. Thus, these results suggest that free E2 concentrations are not affected by physiological concentrations of FFA.

Effect of high-fat intake on lactogenic hormone bioactivity in premenopausal women.

To determine if dietary fat affects the bioactivity of circulating prolactin and growth hormone (lactogenic hormones), a controlled feeding study was conducted in six premenopausal women over two complete menstrual cycles. After an initial 7-10 d dietary adjustment, subjects were randomly assigned to either low-fat (25% of calories) or high-fat (46%) diets. The diets were similar in proportions of energy, protein, P:S ratio, and cholesterol. All subjects completed both diets in a crossover experimental design. Fasting blood samples were collected on alternate days throughout the study. Plasma hormone bioactivity was assayed by the Nb2 lymphoma cell method. Luteal-phase lactogenic hormone bioactivity appeared to be higher (averaging 28%; p less than 0.07) in response to high- rather than low-fat diets. No differences were seen in levels of radioimmunoreactive prolactin or growth hormone. Substantial changes in short-term fat intake may alter the level of lactogenic hormone bioactivity in young women.

Increased plasma pyridoxal 5'-phosphate and vitamin B6 in male adolescents after 4500-meter run.

To investigate the effect of running on vitamin B6 metabolism, plasma pyridoxal 5'-phosphate and total vitamin B6 levels were determined in male adolescent trained athletes before and immediately after a 4500-m run. Six to seven subjects were studied twice during the fall and once during the spring training seasons. A mean increase in pyridoxal 5'-phosphate of 0.77 +/- 0.31 to 1.83 +/- 0.77 nmol/100 ml of plasma was observed after the three runs (p less than 0.01). Similarly, significant increases (p less than 0.05) in plasma total vitamin B6 were seen in all three runs. These increases were not solely due to changes in plasma volume. From urine samples collected on two runs, urinary 4-pyridoxic acid excretion in this group of adolescents was found to be similar to that reported for male adults. While changes in plasma glucose were not significantly correlated with changes in vitamin B6 metabolites, it is hypothesized that the significant changes in vitamin B6 metabolites found in this study were related to an increased need for cofactor for gluconeogenesis. The changes found in this study do not provide evidence for or against an increased need for vitamin B6 in persons that exercise, but does show, for the first time, that exercise in the form of long distance running dramatically alters plasma levels of pyridoxal 5'-phosphate.

Vitamin B-6 status and bioavailability in vegetarian women.

It has been hypothesized that the vitamin B-6 status of vegetarians and nonvegetarians may differ in relation to bioavailability of vitamin B-6. Fasting blood samples and 24-h urine collections were obtained from 13 Seventh-day Adventist (SDA) vegetarian and 16 non-SDA nonvegetarian women aged 50-83 y. The two groups were further subdivided into vitamin users and nonusers. Dietary intake was estimated from a 3-d diet record. Plasma pyridoxal 5'-phosphate (PLP) was measured by an enzymatic method. Vitamin B-6 intakes were similar and provided 85% of the RDA for both groups. The vegetarians consumed significantly more crude fiber than the nonvegetarians. No significant differences were found between the two groups for plasma PLP, urinary 4-pyridoxic acid, and urinary vitamin B-6 among vitamin nonusers or for age categories within groups. There appeared to be no adverse effect of fiber on the availability or metabolism of vitamin B-6 between these free-living groups.

Effect of high dose ascorbic acid on vitamin B6 metabolism.

The influence of ascorbic acid intake on vitamin B6 metabolism in humans was determined in three separate studies. Five subjects in study I and two subjects in study II received 0.5 ( 2 days) and 1 g (7 days) doses of L-ascorbic acid (AA), respectively, before supplementation with 2 mg of pyridoxine hydrochloride (PN-HCl). From daily diet records the percentage of the intake of vitamin B6 excreted as urinary 4-pyridoxic acid (4PA) was calculated. Because of the variability in vitamin B6 intake, study III was conducted with four male and four female subjects, who received identical meals on days 2, 3, 9, and 10 and 1 g of AA on days 4 to 10. Two milligrams of PN.HCl were given the day before and the last day of AA administration. In contrast to studies I and II, where excretion of 4PA appeared to increase on the day of PN.HCl supplementation plus AA compared to pre-AA, urinary 4PA excretion in the eight subjects of study III was not significantly different when the same PN.HCl supplemented pre-AA and post-AA days were compared. Also, the fasting plasma pyridoxal 5'-phosphate level was not significantly altered when AA was administered. It is concluded that short-term AA supplementation did not alter vitamin B6 metabolism.

Selenium status of vegeterians, nonvegetarians, and hormone-dependent cancer subjects.

Human blood selenium (Se) levels have been related to the types of food consumed, bioavailability of Se, and various disease states, including cancer. Some of these interrelationships were investigated in this study in Corvallis, OR (a low soil-Se region) using adult vegetarian and omnivorous subjects, some of whom had hormone-dependent cancer. The study groups were comprised of 48 Seventh-day Adventist vegetarians, 16 Seventh-day Adventist nonvegetarians, 52 non-Seventh-day Adventist nonvegetarians, and 16 nonvegetarian hormone-dependent cancer subjects. Fasting blood samples and 3-days dietary intake information were obtained from each subject. Whole blood Se levels, measured fluorimetrically, correlated positively with dietary protein, riboflavin, niacin, and oleic and linoleic acids but not with 11 other nutrients. Due to limited literature values, dietary Se could not be assessed. There was no significant difference in blood Se values between the four groups (which ranged from 0.069 microgram Se/ml for Seventh-day Adventist nonvegetariants to 0.112 +/- 0.050 microgram Se/ml for non-Seventh-day Adventists nonvegetarians and nonvegetarian hormone-dependent cancer patients). All values were well below averages reported for other regions of the United States. These data suggest a relationship between blood Se and the consumption of meat, milk, and cereal products, but it is not simply a difference between the vegetarian and nonvegetarian diets.

Dietary and hormonal interrelationships among vegetarian Seventh-Day Adventists and nonvegetarian men.

The relationship between dietary nutrients and plasma testosterone, 5 alpha-dihydrotestosterone, estradiol-17 beta, luteinizing hormone, and prolactin levels was investigated in 12 Seventh-Day Adventist (SDA) vegetarian (SV), 10 SDA nonvegetarian (SNV), and 8 non-SDA nonvegetarian (NV) men. Fasting blood samples and 3-day dietary intake information were obtained from each subject. The SV subjects consumed significantly more crude and dietary fiber than the SNV and NV subjects, respectively. Plasma levels of testosterone and estradiol-17 beta were significantly lower in the SV than in the omnivores. Additionally, the plasma levels of testosterone and estradiol-17 beta of the combined groups (SV, SNV, and NV) revealed a significant negative relationship with their crude and dietary fiber intakes. These subjects hormonal milieu was related to specific dietary constituents, possibly leading to a decreased plasma concentration of androgen and estrogen in vegetarians. Implications include the possible modification of prostate cancer risk through dietary intervention.

Effect of low- and high-fat intakes on the hormonal milieu of premenopausal women.

To compare the influence of low- and high-fat diets on hormone metabolism, we studied six premenopausal women over two complete menstrual cycles. After an initial 7-10 d dietary adjustment subjects were randomly assigned to either low- (25% of calories) or high-fat (46% of calories) diets. The diets were isocaloric and similar in proportions of protein, polyunsaturated:saturated fatty acid ratio, fiber, and cholesterol. All subjects completed both diets in a crossover experimental design. Fasting blood and 24-h urine samples were collected every other day. No significant differences in plasma luteinizing hormone, progesterone, prolactin, estrone (E1), and estradiol-17 beta (E2) or in urinary E1, E2, and estriol were observed. Prolactin secretion in response to thyrotropin-releasing hormone stimulation was also unchanged. Dietary fat may influence breast-cancer incidence by modulating the hormonal environment; however, an increase in short-term fat intake from 28 to 51 g/1000 kcal did not lead to substantial differences in the hormonal milieu.

Dietary and hormonal evaluation of men at different risks for prostate cancer: fiber intake, excretion, and composition, with in vitro evidence for an association between steroid hormones and specific fiber components.

Dietary fiber intake and fecal fiber excretion were investigated in 53 Seventh-day Adventist men: 18 nonvegetarians (NVs), 20 lactoovovegetarians (LOVs), and 15 vegans (Vs). Three-day composite diets and stools were analyzed for neutral detergent fiber (NDF), hemicellulose, cellulose, lignin, and pectin. In vitro binding of estrone (E1), estradiol-17 beta (E2), and testosterone (T) to a water-insoluble fiber fraction obtained from these diets was correlated with the intake of specific dietary fiber components. Vs consumed and excreted significantly more of all fiber components than did LOVs or NVs. LOVs consumed more of all fiber components (except cellulose) than did omnivores and excreted more NDF, hemicellulose, and cellulose. Dietary lignin was positively correlated with T binding in the V group. There were significant relationships for all groups combined between lignin and water-insoluble fiber binding of E1, E2, and T. Further study is needed to clarify relationships between fiber components, steroid-hormone metabolism, and risk of prostate cancer.

Urinary, plasma, and erythrocyte carnitine concentrations during transition to a lactoovovegetarian diet with vitamin B-6 depletion and repletion in young adult women.

The purpose of the present study was to determine whether vitamin B-6 depletion and repletion influence carnitine concentrations in the plasma, erythrocytes, and urine of young adult women consuming a low-carnitine (30 micromol/d), lactoovovegetarian diet. Doses of vitamin B-6 were administered sequentially as follows: 1.60 mg/d during adjustment for 9 d, 0.46 mg/d during depletion for 27 d, 1.26 mg/d during the first repletion for 21 d, 1.66 mg/d during the second repletion for 21 d, and 2.06 mg/d during the third repletion for 14 d. Urinary carnitine tended to decline but was not significantly different throughout the 92-d study period. Plasma short-chain acylcarnitine and total carnitine decreased during vitamin B-6 depletion; however, the ratio of plasma acylcarnitine to total carnitine was not significantly different during changes in vitamin B-6 intake. Erythrocyte long-chain acylcarnitine increased during vitamin B-6 depletion, but the ratio of erythrocyte acylcarnitine to total carnitine did not respond to changes in vitamin B-6 intake. Plasma free and total carnitine concentrations were only weakly correlated with plasma pyridoxal-P concentration (r = 0.28 and r = 0.29, respectively; P < 0.01). No significant correlations were observed between urinary carnitine excretion or erythrocyte carnitine concentrations and plasma or erythrocyte pyridoxal-P concentrations. Thus, a vitamin B-6 intake of 0.5 mg/d does not affect carnitine concentrations in biological fluids and therefore is unlikely to affect endogenous carnitine synthesis over 27 d. The changes in carnitine indexes that we observed were probably due to adaptation to a vegetarian pattern of exogenous carnitine consumption.

Plasma and urine taurine levels in vegans.

Plasma taurine levels and urinary taurine excretion were measured in 12 strict vegetarian (vegan) males who had maintained a vegan diet for 53 +/- 26 mo (SD) and in 14 male nonvegetarian control subjects. Plasma taurine levels differed (45 +/- 7 vs 58 +/- 16 mumol/L, respectively). Urinary taurine excretion was lower (266 +/- 279 vs 903 +/- 580 mumol/d), urinary N pi-methylhistidine was barely detectable, and urinary N tau-methylhistidine was significantly reduced (296 +/- 87 vs 427 +/- 19 mumol/d) in the vegans. Analysis of 3-d dietary diaries kept by the vegans indicated marginal to adequate intake of protein, carbohydrate, vitamin B-6, methionine, and cystine; inadequate intake of zinc; and negligible intake of taurine. Prolonged absence of dietary taurine intake causes decreased plasma taurine and severely restricted urinary taurine output.

Dietary and hormonal evaluation of men at different risks for prostate cancer: plasma and fecal hormone-nutrient interrelationships.

Relationships between dietary nutrients and plasma and fecal estrone, estradiol-17 beta, testosterone, and plasma prolactin concentrations were studied in young Seventh-day Adventist men: 18 nonvegetarians (NVs), 20 lactoovovegetarians (LOVs), and 15 vegans (V). Blood samples and 3-d dietary records were obtained. Contemporaneously collected diet composites and stool samples were analyzed for fiber. Vs and LOVs consumed significantly more fiber than did the omnivores, whereas NVs and LOVs consumed more saturated fatty acids than did Vs. Although plasma steroid-hormone status did not differ, Vs had significantly higher fecal estrogen concentrations than did NVs or LOVs. Plasma prolactin concentrations were significantly higher in NVs and LOVs than in Vs. Significant relationships were observed for the combined groups between dietary and fecal fiber components and fecal, but not plasma, steroid hormones. For the combined groups, prolactin concentrations were positively correlated with saturated fatty acid intake. Further research on the effects of dietary nutrients on endocrine homeostasis in other age groups is warranted.

The relationship among adiposity, diet, and hormone concentrations in vegetarian and nonvegetarian postmenopausal women.

The relationships among anthropometric variables, dietary nutrients, and plasma steroid, polypeptide, and binding-protein hormone concentrations were investigated in 24 Seventh-day Adventist postmenopausal women, 12 vegetarian (SV) and 12 nonvegetarian (SNV). Fasting blood and 7-d dietary intake information were collected. SVs consumed significantly more crude and dietary fiber and fewer saturated fatty acids than did SNVs. The thigh and sum of three skinfold-thickness measurements were significantly greater for SNVs than for SVs. Plasma concentrations of estradiol-17 beta were significantly lower in SVs than in SNVs. Significant relationships were observed for the combined groups (SV and SNV) between estradiol-17 beta and triceps and suprailiac skinfold thickness and body fat. Plasma concentrations of estradiol-17 beta of the combined groups revealed a significant negative relationship between their crude and dietary fiber intakes. Further study delineating the effects of adiposity and dietary nutrients on basal concentrations of sex hormones is warranted.

Dietary and hormonal interrelationships in premenopausal women: evidence for a relationship between dietary nutrients and plasma prolactin levels.

To investigate possible differences in tissue exposures to reproductive hormones and to determine hormone-nutrient interrelationships, we studied 10 vegetarian and 10 nonvegetarian premenopausal Seventh-day Adventist women. Over 3 d in each of three consecutive menstrual cycles, we collected diet records, fasting midluteal phase blood, and 24-h urine samples. During each study period, we measured plasma and urinary estrogens, plasma free and protein bound-estradiol-17 beta, the binding capacity of sex-hormone-binding globulin (SHBG), androgens, progesterone, and prolactin levels. The omnivores consumed significantly more protein, total and saturated fatty acids, oleic and linoleic acids, and cholesterol than did the vegetarians. Hormonal status and binding capacity of SHBG were similar in both groups. However, for nonvegetarians, prolactin levels were positively correlated with dietary energy, protein, total and saturated fatty acids, and oleic acid. Further study delineating the effects of specific dietary nutrients on the basal level of prolactin secretion is warranted.

Vitamin B-6 requirement and status assessment of young women fed a high-protein diet with various levels of vitamin B-6.

The vitamin B-6 requirement of young women consuming a constant high-protein diet (1.55 g/kg body wt) and the effect of various ratios of vitamin B-6 to protein on this requirement were studied. Eight women were fed a lactoovovegetarian basal diet containing 0.45 mg vitamin B-6 (2.66 micromol as pyridoxine) and 30 micromol carnitine for 92 d. The protocol consisted of successive baseline adjustment (9 d), depletion (27 d), and repletion (two 21-d and then one 14-d) periods. Vitamin B-6 intakes were 1.60, 0.45, 1.26, 1.66, and 2.06 mg, resulting in ratios of vitamin B-6 (in mg) to protein (in g) for the five periods of 0.016, 0.005, 0.013, 0.017, and 0.021, respectively. Direct and indirect as well as short- and long-term vitamin B-6 status measures were assessed weekly. Regression analysis revealed that the amount of dietary vitamin B-6 required to normalize urinary 4-pyridoxic acid, plasma pyridoxal-P, erythrocyte pyridoxal-P and pyridoxal, and erythrocyte alanine and aspartate aminotransferase activity coefficients to predepletion baseline values was 1.94 mg vitamin B-6/d (0.019 mg vitamin B-6/g protein). This study suggests that the current vitamin B-6 recommended dietary allowance of 1.6 mg/d based on 0.016 mg/g protein is not an adequate intake and may require reevaluation.

Safflower oil consumption does not increase plasma conjugated linoleic acid concentrations in humans.

Conjugated linoleic acid (CLA) is a mixture of positional and geometric isomers of linoleic acid (LA) with conjugated double bonds. CLA has anticarcinogenic properties and has been identified in human tissues, dairy products, meats, and certain vegetable oils. A variety of animal products are good sources of CLA, but plant oils contain much less. However, plant oils are a rich source of LA, which may be isomerized to CLA by intestinal microorganisms in humans. To investigate the effect of triacylglycerol-esterified LA consumption on plasma concentrations of esterified CLA in total lipids, a dietary intervention (6 wk) was conducted with six men and six women. During the intervention period a salad dressing containing 21 g safflower oil providing 16 g LA/d was added to the subjects' daily diets. Three-day diet records and fasting blood were obtained initially and during dietary and postdietary intervention periods. Although LA intake increased significantly during the dietary intervention, plasma CLA concentrations were not affected. Plasma total cholesterol and LDL-cholesterol concentrations were significantly lower after addition of safflower oil to the diet. In summary, consumption of triacylglycerol-esterified LA in safflower oil did not increase plasma concentrations of esterified CLA in total lipids.

Inhibitory effect of conjugated dienoic derivatives of linoleic acid and beta-carotene on the in vitro growth of human cancer cells.

The effects of physiologic concentrations of conjugated linoleic acid (CLA) and beta-carotene were assessed on human (M21-HPB, malignant melanoma; HT-29, colorectal; MCF-7, breast) cancer cells. The incubation of cancer cells with CLA showed significant reductions in proliferation (18-100%) compared to control cultures. M21-HPB and MCF-7 cell mortality was dose- and time-dependent. beta-Carotene was inhibitory to breast cells only. MCF-7 cells supplemented with CLA incorporated significantly less [3H]leucine (45%), [3H]uridine (63%) and [3H]thymidine (46%) than control cultures. M21-HPB and HT-29 cells supplemented with CLA incorporated less [3H]leucine (25-30%). These in vitro results suggest that CLA and beta-carotene may be cytotoxic to human cancer cells in vivo.

Modulation of MCF-7 breast cancer cell signal transduction by linoleic acid and conjugated linoleic acid in culture.

The effects of modifying membrane fatty acid composition on cell growth, phospholipase C (PLC) and protein kinase C (PKC) activities, and prostaglandin E2 (PGE2) secretion were investigated. Hormone responsive MCF-7 human breast cancer cells were incubated in a serum-free medium containing epidermal growth factor and supplemented with physiologic concentrations (0.18-1.78 x 10(-5) M) of linoleic acid (LA) or conjugated linoleic acid (CLA). Linoleic acid stimulated cancer cell growth, while CLA was inhibitory. Supplementation with LA or CLA altered cell membrane composition. Linoleic acid stimulated PLC activity with or without GTP gamma (S), and tended to increase membrane PKC activity. However, CLA supplementation did not modify membrane PLC or PKC activity. Prostaglandin E2 secretion was not influenced by LA or CLA. These data show that growth inhibition by CLA was not mediated through PLC-, PKC- or PGE2-dependent signal transduction pathways, suggesting that another inhibitory mechanism may be involved. Although biological differences appeared to be modest (5-20% of control), the fact that LA and CLA treatment resulted in significant biological effects at physiologic concentrations is relevant, since most human cancers require years to develop.

Proliferative responses of normal human mammary and MCF-7 breast cancer cells to linoleic acid, conjugated linoleic acid and eicosanoid synthesis inhibitors in culture.

Potential mechanisms for the stimulation or inhibition of cell growth by linoleic acid (LA) and conjugated linoleic acid (CLA) were investigated by using eicosanoid synthesis inhibitors. Normal human mammary epithelial cells (HMEC) and MCF-7 breast cancer cells were incubated in serum-free medium supplemented with LA or CLA and cyclooxygenase (indomethacin; INDO) or lipoxygenase (nordihydroguaiaretic acid; NDGA) inhibitors. Linoleic acid stimulated the growth and [3H]thymidine incorporation of normal HMEC and MCF-7 cancer cells, while CLA was inhibitory. Supplementation with LA increased intracellular lipid peroxide concentrations in normal HMEC and MCF-7 cancer cells, whereas CLA did not affect lipid peroxide formation. Normal HMEC and MCF-7 cells supplemented with LA and INDO or NDGA resulted in growth inhibition. The treatment of normal HMEC with CLA and INDO or NDGA, and MCF-7 cells with CLA and INDO stimulated cell growth. However, the addition of CLA and NDGA to MCF-7 cells resulted in synergistic growth suppression suggesting that CLA effects were mediated through lipoxygenase inhibition. Although NDGA was more inhibitory of cell growth in the presence of LA or CLA than INDO, growth was associated with both prostaglandin and leukotriene production. Additional studies are warranted to elucidate the mechanism(s) whereby LA or CLA affect breast cell growth.