Duration: 10/01/2002 to 09/30/2008

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

There is a well-established connection between diet and health. Spiraling health care costs have catalyzed a change in emphasis toward preventing chronic diseases (e.g., heart diseases, cancer, osteoporosis) instead of treating them once they develop. Growing evidence indicates that increased intakes of vitamins A, C, and E, the B-vitamins, carotenoids, calcium, selenium, magnesium, zinc, chromium, phytochemicals and foods rich in these and other nutrients may help prevent such diseases and improve the health of Americans. These purported benefits have led to the widespread consumption of these nutrients in the form of diet supplements (e.g., vitamins, minerals, botanicals and other phytonutrients) and the emphasis on increased intake of fruits, vegetables and grains.

Almost 70% of Americans use a complementary or alternative medicine therapy during their lifetime (1). Herbal and phytonutrient supplements induce phase I (2)and phase II (3)enzymes which may alter the absorption, distribution, metabolism and elimination (ADME) of essential nutrients. For example, both vitamins E (4) and K (5)are known to be altered by ingredients in phytonutrient supplements.

In 1998, United States sales of botanical/herbals were 3.9 billion of the $13.7 billion dietary supplement market (6). Yearly growth in the dietary supplement market has been estimated to be about 6.8 % (7). In 1998, the leading botanicals were St. John's wort ($45 million); ginkgo biloba ($40 million); echinacea ($38 million); saw palmetto ($29 million); valerian ($20 million) and kava kava ($19.5 million) (6). In 2000, glucosamine was the leader with sales of $402 million. Homeopathic compounds followed: essential fatty acids ($182 million); S-adenosyl methionine (SAMe ) at $170 million and probiotics at $111 million (836}. Obviously, the American public is speaking loudly with its pocketbook. Of interest and potentially of concern is the trend of wanting combinations of products in America in contrast to the more common use of standardized extracts of a single herbal in Germany (6).

However, because botanicals are considered to be food in the United States (US), they do not undergo the extensive research required for drugs. Thus, their efficacy is sometimes questioned. In most cases, it is not known if they can be absorbed.. More importantly, however, is the lack of knowledge of their affect on the absorption and utilization of other essential nutrients. This deficit in knowledge will be addressed, at least in part, by the research being proposed here. It is well established that foods, food constituents, nutrients, exercise, physiological status, and maturity can alter nutrient bioavailability to the point of affecting health. Progress has been made toward understanding factors that affect nutrient bioavailability for humans. However, because of the upsurge in use of non traditional food sources and botanicals, it is critical to know if the phytochemicals in such sources can be absorbed and/or how these foods and botanicals affect nutrient absorption in general. Information is needed about how they affect absorption of under consumed nutrients and nutrients which are known to reduce the risk of the major causes of death (cardiovascular diseases and certain cancers) and disability (osteoporosis). Failure to gather such information increases the potential that the incidence of conditions which have been improving may start to rise.

Aside from the bioavailability of bioactive components of botanicals and the effect of these components on essential nutrients, there is also the issue of absorption of important nutrients that are being added as fortification to foods. These include foods in which the nutrient is normally present (e.g., calcium to cheese) and those which are not inherently a good source of the nutrient (e.g., calcium in orange juice and rice).

Because of the interactions and collaborations of W-143 scientists, agriculture- and health-related entities have been provided with considerable information for use in the prevention of nutrition-related chronic diseases, consistent with federal priorities (8). Data related to previous priorities addressed in W-143 research has served, in part, as the basis for

* the fortification of cereal grains with folate to decrease the risk of neural tube defects;

* the inclusion of health statements about calcium and osteoporosis, and hyperhomocystinemia and heart disease on food labels; and

* the increased DRIs for calcium for young adults.

Without question, the information derived from the proposed research and the proposed dissemination of information to both the scientific community and lay public will provide part of the framework on which future nutrient recommendations can be based.

Related, Current and Previous Work

Phytonutrients: Isoflavones. Osteoporosis is a bone disease characterized by reduced bone mass leading to fractures. Nationwide, approximately 1.5 million fractures per year are attributed to osteoporosis (9). Direct health care costs for these fractures are estimated at $13.8 billion per year nationally in 1995 dollars (10). The projected increase in osteoporotic fractures over the next 60 years is 300%.

Osteoporosis is more prevalent among women, partially because of the accelerated rate of bone resorption compared to bone formation with menopause. Estrogen Replacement Therapy (ERT) is a mainstay for the prevention of osteoporosis in postmenopausal women. ERT stabilizes and even increases axial bone, particularly in women with low bone mineral density (BMD) (11). ERT is more effective in increasing BMD in postmenopausal women with calcium supplementation to 1 g/day than usual calcium intakes of 563 mg/d (11). When both calcium to achieve 1 g/day and 25-OH vitamin D to achieve serum levels of 75 nmol/L were also given, even low-dose (0.3 mg/d) continuous estrogen and progesterone (2.5 mg/d) increased spinal BMD by 3.5% and by 5.2% among patients who were at least 90% compliant compared to controls (12).

ERT is an antiresorptive therapy and reduces bone turnover to premenopausal levels for as long as therapy continues. Both rates of bone formation and resorption decrease in response to ERT, but the effect on resorption is dominant; thus, BMD is stabilized, or in some cases such as when calcium intake is adequate, increases (12). However, for various reasons, there is some reluctance and/or medical inappropriateness to use estrogen. Given the resistance to the use of ERT, and its contraindication in women at risk of breast cancer, there is an escalating body of research into plant phytoestrogens as a possible alternative. Phytoestrogens are compounds, with structural or functional similarity to estradiol, found in a range of plant foods (13). The three main classes are isoflavones (soybean products, chickpeas and other legumes); ligands (oilseeds, principally flaxseed), cereal bran and legumes; and coumestans, (alfalfa and clover) (14). The major soy isoflavones, genistein and daidzein, possess weak estrogenic activity and lower affinity than 17-B estradiol for an estrogen receptor (ER) on osteoblasts, yet estrogen down-regulates activity of osteoclasts, thereby limiting bone resorption (15). The exact mechanism is as yet unclear. Phytoestrogens have higher affinity for ER-B, which is present in bone, brain, bladder, and vascular epithelia, than ER-A. Phytoestrogens may have effects through the ER, but also through unrelated events (13)such as genistein inhibiting tyrosine protein kinases, DNA topoismerase I and II and ribosomal S6 kinase.

Phytoestrogens also demonstrate antiestrogenic activity, for example in the breast (16). This raises the possibility that they may be acting like SERMs (selective estrogen receptor modulators), such as tamoxifen and raloxifene, than estrogen, i.e. an estrogen agonist-antagonist.

The limited amount of data on the skeletal effects of isoflavones suggest agonistic effects. Ecological studies show a lower incidence of osteoporosis among Asian populations consuming high soy diets (20-50 mg/d ) (17)compared to Western populations (13), although the limitations of such studies are acknowledged. Ipriflavone, a synthetic isoflavone derivative, has been reported to inhibit bone loss in various experimental models. In vitro work has shown that ipriflavone (18)and coumestrol (19)inhibit calcium release from bone cell culture. Studies using the ovarectomized rat model have shown comparable bone sparing effects of 17b-estradiol and soybean meal (20). Anderson et al. (21)demonstrated a biphasic response of genistein on ameliorating bone loss in lactating, ovarectomized rats, suggesting estrogen agonistic action only at low doses. Coumestrol, but not red clover extract, decreased bone loss at the spine, femur, and whole body in ovarectomized rats (22).

Isoflavones occur in concentrated amounts in legumes, predominantly as the glycoside conjugates. Upon ingestion, isoflavones are biotransformed by intestinal microflora for absorption. Isoflavones are hydrolyzed by glucosidases to the aglycones, daidzein, genistein and glycetein. Isoflavones as the glycosides are more bioavailable than the aglycones, perhaps due to reduced degradation in the presence of a sugar moiety. Phytoestrogens are more likely to be free to bind with an ER than estrogens because they bind with 10-fold less affinity to sex hormone binding globulin (SHBG) (23).

Saponins. There is great interest in saponins as phytonutrients because of their pharmaceutical action. Using a variety of approaches, saponins have been shown to have anticancer, anti-inflammatory, antivirus, antiulcer and various other activities (24). Saponins are amphiphilic compounds which interact with both lipid and water soluble compounds. They have either triterpenoidal or steroidal structures and are found in greatest concentrations in legumes (25). Soybeans are one of the richest food sources of saponins, but other edible beans and peas also contain considerable quantities of the same saponins. Most of the soybean extracts available on the market today contain both isoflavones and saponins and it is quite likely that some of the biological actions attributed to isoflavones are actually due to saponins. Undesirable effects from saponin consumption can occur also. For example, glycyrrhetic acid, a triterpenoid saponin, alters the renin-aldosterone system which results in low blood potassium and hypertension (26).

Lignins. Lignins are a group of phytoestrogens with possible cancer protective properties (27). However, furofuran lignins (sesamin and sesaminol from sesame) inhibit cytochrome P450 CYP3A. and alter the metabolism and excretion of tocopherols (vitamin E). Inhibition of CYP3A leads to accumulation of tissue tocopherols, but the long term ramifications of altered tocopherol metabolism by plant lignins are not known. Other plant flavonoids including isoflavones alter cytochrome P450 enzymes (2). Since genistein is a hydroxylated flavonoid and high concentrations of hydroxylated flavonoids inhibit CYP3A (28), it possible that isoflavones may alter tocopherol metabolism also.

Other. The literature clearly establishes that: 1) many phytonutrients induce an apoptotic (programmed cell death) response in a variety of cancerous human cell lines; and 2) this effect appears to be limited to cancerous cells. Thus some of the beneficial impact of phytochemical ingestion may result from enhanced apoptosis, which could limit or reverse growth of human cancers.

Various flavonoids induce apoptosis, which is the adaptive and regulated process widely utilized in mammalian tissues to limit or reverse excess cellularity. Genistein (29)and possibly lycopene (30)drive apoptosis in prostate cancer cells. Epigallocatechin 3-gallate (EGCG) (31), flavone (32), and various indoles and isothiocyanates (33), including sulforaphane (34), induce apoptosis in colon cancer cells. Lutein induces apoptosis in transformed human mammary cells (35). Phytonutrients stimulate apoptosis in several cancers of blood cells, including resveratrol (36)as well as indoles and isothiocyanates (37) in leukemic cells, curcumin in macrophage tumor cells (38)and EGCG in monocytes (39). Stomach cancer cell lines respond to EGCG (40)and resveratrol (41)by becoming apoptotic. Beta-sitosterol induces apoptosis in various cancerous cell lines (42). The apoptotic effect of EGCG (43)is dose-dependent and specific for cancerous but not normal cell lines, as is the case for lutein in mammary cancer cells (35).

Carnosine is a beta-alanine-histidine dipeptide found in skeletal muscle. Early reports have shown that dietary carnosine is absorbed into plasma intact through a specific transport system in the small intestine (44). However, kinetics of absorption, dose response and the half-life of plasma carnosine, as well as antioxidant properties, are not known.

Minerals and Vitamins: Calcium. Dairy products supply 72 % of the calcium in the adult US diet, grain products about 11 %, and vegetables and fruits about 6 % (45). It is difficult for most people to ingest sufficient calcium from foods in a cereal-based diet without liberal consumption of dairy products. Many have turned to dietary supplements to meet their calcium needs. In response, food manufacturers have developed calcium-fortified products (e.g., orange juice; rice; breads; low or non caloric beverages) that have had a range of market success.

Although calcium absorption efficiency varies inversely with load, its fractional absorption from various dairy products is similar at ~30 % (46). The calcium from most supplements is absorbed as well as that from milk, as solubility of salts at neutral pH has little impact on calcium absorption (47). Absorption of a very soluble salt, calcium-citrate-malate (CCM), is better than other salts (48).

Several plant constituents reduce the absorption of calcium from these sources. The most potent inhibitor is the oxalic acid that occurs in high concentrations in spinach, rhubarb and, to a lesser extent, in sweet potatoes and dried beans (49). Capsaicin containing compounds in chile have laxation effect which increases fecal excretion of calcium(50). Enhancers of calcium absorption are not well-characterized.

Calcium excretion and retention are influenced by dietary sodium, age, and race. Urinary calcium losses account for 50% of the variability in calcium retention (51). Urinary calcium losses are most affected by protein, caffeine, and sodium intake. Because sodium and calcium share some kidney transport systems, each increment of sodium excreted pulls calcium out with it (52). Shortt et al.(53)indicated in adult women a gram of extra sodium would result in an additional rate of bone loss of 1%/ year if urinary calcium lost came from the skeleton. Moreover, urinary calcium loss does not appear to be corrected by a compensatory increase in calcium absorption or reduced endogenous fecal secretion, despite high sodium resulting in increased 1,25 (OH)2 vitamin D levels (54).

During growth, calcium retention is high. Average calcium retention in white female adolescents of ~300 mg/day in contrast to ~0 mg/day in women just a few years older at peak bone mass has been measured (55). After menopause calcium balance is typically negative (56). It is not clear whether racial differences in calcium metabolism in response to dietary salt occur at other life stages because it has only been studied in children.

Men have greater bone mass than women, their vertebrae have larger diameters, and their trabecular bone remains better preserved. To achieve that greater bone mass, peak calcium accretion rates are higher in boys than girls (peak bone mineral content velocity was 282 mg/d in boys vs 212 mg/d in girls determined by cross-sectional analysis of Canadian children), although it occurred 1.5 y later in boys (aged 14.5 y for boys vs. 13 y for girls) (57). The difference in calcium accretion between girls and boys increased through puberty.

Iron. Iron is a necessary micronutrient; deficiency results in anemia, pallor, weakness, fatigue, dyspnea, palpitations, sensitivity to cold, oral cavity and gastrointestinal tract abnormalities, and reduced capacity for work. Due to an increased demand for iron during times of rapid growth or iron loss (e.g., infancy, adolescence, pregnancy, and menstruation), these groups experience a higher incidence of iron deficiency than the general public.

Although iron is an essential nutrient, legitimate concerns have been raised about its possible role in catalyzing oxidation. Such catalysis has been postulated to increase oxidized LDL and ultimately lead to increased risk of heart attacks.

Several reviews have summarized knowledge of iron bioavailability (58). Human studies have indicated that many nutrients and dietary constituents, exercise, physiological status, and maturity alter iron bioavailability such that iron status is increased or decreased.

Vitamin B6. Vitamin B6 occurs in foods as pyridoxine, pyridoxamine, pyridoxal, as the phosphorylated forms of these three compounds, and as a conjugated form commonly referred to as pyridoxine glucoside. Vitamin B6 is absorbed primarily from the jejunum (59) and transported to the liver where it undergoes conversion to pyridoxal 5'-phosphate (PLP) (60). Also, in the liver, pyridoxal is converted to 4-pyridoxic acid, the major urine metabolite. Pyridoxal 5'-phosphate is released from liver and circulates in blood bound to albumin.

Vitamin B6 functions in the metabolism of amino acids, lipids, carbohydrates, in niacin formation, in erythrocyte metabolism and function, in hormone modulation, and in the development and maintenance of a competent immune system (61). These functions illustrate its importance in health and the need to understand those factors, e.g., bioavailability, that determine its status.

Several studies have noted suboptimal vitamin B6 status and cardiovascular disease. In studies comparing vitamin B6 status in coronary disease patients with controls, low vitamin B6 intake and/or plasma PLP were found to be an independent risk factor for cardiovascular disease (62). Rimm and colleagues (63) from the Nurses Health Study noted a significant inverse relationship between dietary intakes of folate and vitamin B6 and mortality and morbidity from cardiovascular disease during a 14-year period.

Several studies found that fasting plasma tHcy (homocysteine) concentrations were not strongly influenced by vitamin B6 depletion, however plasma tHcy levels after a methionine load were increased in B6 depleted subjects (64). This suggests that fasting concentrations are more influenced by remethylation (folate and B12) and post-methionine load concentrations are more influenced by transsulfuration (B6). Ubbink et al. (65)also reported that plasma tHcy concentrations after a methionine load were reduced in vitamin deficient asthma patients who were supplemented with B6. The use of a dietary challenge, specifically a methionine load, suggests that in vitamin B6 deficiency, homocysteine metabolism is impaired.

The principal function of folate coenzymes is to accept or donate one-carbon units in key metabolic pathways. Knowledge of vitamin B6 regulation of one-carbon metabolism with respect to PLP dependent serine hydroxymethyl transferase (SHMT) activity in tetrahydrofolate (THF) metabolism is limited (66). Martinez et al. (67) found that dietary vitamin B6 deficiency in rats caused significantly reduced plasma and liver PLP, and a 41% lower SHMT activity, indicating pathway disruptions. Thus, such research is needed.

Folate. Folate functions in transporting single carbon fragments in the synthesis of nucleic acids and in the metabolism of amino acids. Folate deficiency leads to impaired cell division that presents as neural tube defects and anemia and to dysfunctional amino acid metabolism that presents as hyperhomocystinemia which is accepted as a major risk factor for heart disease.

Mono and short chain polyglutamyl folates are absorbed from the intestine directly. Polyglutamyl forms must first be hydrolyzed to the monoglutamyl and short chain polyglutamyl forms before absorption. The capacity to hydrolyze polyglutamyl folates far exceeds the capacity to transfer folate from intestine to blood (68). Current estimates of the efficiency of dietary folate absorption are variable depending on the experimental protocols used (69).

B-carotene and Vitamin A. B-carotene protects against oxidative stress and heart disease by quenching singlet oxygen and enhancing the immune response. Humans derive most of their vitamin A from B-carotene (70). Because antioxidants have a substantial potential to minimize oxidative events, B-carotene is considered to be biologically and clinically important (71). Despite its key physiologic roles, its widespread use as a dietary supplement, and regular use in clinical trials, the dynamics of B-carotene metabolism are unknown.

A suitable animal model that mimics B-carotene metabolism of humans is not available and use of radioactive B-carotene to study absorption in humans can only be justified in special cases. Progress in understanding the kinetics of B-carotene metabolism in humans has been hindered by a scarcity of stable isotope labeled carotenoids and by analytical challenges in measuring them in biologic tissues. However, recent successes of W143 researchers and others in the synthesis and analysis of B-carotene-d8 (72-74)has provided the ability to study the kinetics of B-carotene absorption and metabolism in humans (75).

From a human health perspective, there are a few central questions concerning carotenoid absorption and metabolism that need to be addressed: (a) Absorption/bioavailability. A number of large-scale intervention studies to determine if B-carotene supplements can reduce the risk of cancer or cardiovascular disease have involved the regular intake of 15 to180 mg B-carotene/d. (b) If B-carotene serves a role apart from its provitamin A function, the pharmacodynamics of the unmodified molecule must be better understood before accurate interpretation of the results of these clinical trials is possible. This need is well illustrated by the Alpha-Tocopherol, Beta Carotene Cancer Prevention Study (76) which indicated that lung cancer increased in male smokers consuming B-carotene. Little data is available concerning human carotenoid absorption, though it is known that the rate and extent of the conversion of dietary carotenes to retinol can vary greatly with intake and species (77).

While the conversion of B-carotene to retinol is widely accepted, quantitative aspects of the conversion and the influence of the many factors that affect it are poorly understood. Giving B-carotene to rabbits increases serum all-trans retinoic acid (78). It remains to be seen if B-carotene produces similar results in humans. Retinoic acid is a key regulatory retinoid (77), a vertebrate morphogen and a human teratogen (79).

CRIS Search: A CRIS search with key bioavailability and phytochemical or phytonutrient revealed 58 active projects. The committee is familiar with all projects There are no similar regional projects.

This proposed project is quite unique because it focuses on the effects of phytonutrients on the ADME of calcium, iron, B-carotene, lutein, and vitamins A, E, B6 and folate. It is also unique because state of the art developments in isotope technology, molecular biology, and biochemistry are used. Labeling nutrients and foods with a variety of isotopes provides information of greatest significance that is almost impossible to obtain with other approaches. A rapid assessment of the overall ADME of a nutrient can only be made in an appropriate time frame by use of appropriate isotope labels. In addition, using a combination of these technologies enables W-143 scientists to study humans, animal models, and cells for a more complete understanding of the ADME of phytonutrients (saponins and isoflavones), calcium, iron, B-carotene, lutein, and vitamins A, E, B6 and folate. The approaches used by the W-143 investigators continue to provide cost-effective and time-efficient information about the ADME of these key nutrients. Past project citations are in Appendix A.

Objectives

1. Determine the ADME of phytonutrients or the impact of phytonutrients, other nutrients or factors on the ADME of essential nutrients.
   
2. Collaboratively develop and maintain a website for dissemination of technical and lay publications related to bioavailability.
   
3. Foster collaboration among stations in the development, validation, standardization, and/or application of technologies, samples, substrates and other resources.
   
4. 
   

Methods

Past W-143 projects indicated that the information regarding nutrient bioavailability is most efficiently acquired by the collaboration of its scientists. The specific questions to be answered by the proposed W-143 project to accomplish the objectives will vary for each of the individual nutrients being studied. As previously indicated, the questions will depend on the current knowledge regarding the bioavailability of each nutrient and any special concerns for human nutrition. The specific collaborative research designs are described below. Potential interactions of AES, ARS, and CES scientists are detailed in Appendix B. Objective 1. Determine the ADME of phytonutrients or the impact of phytonutrients, other nutrients or factors on the ADME of essential nutrients Soy isoflavones. The UC Davis station will characterize the ADME of soy isoflavones over time after administering a single dose of soy protein isolate (100 mg total isoflavones) to humans. Blood will be analyzed for total genistein and diadzein by HPLC. From the area under the plasma concentration curve (AUC), the relative bioavailability, and half lifes (t1/2) for absorption (t1/2a), distribution (t1/2d), and elimination (t1/2e), and the residence time (RT) of the isoflavones will be calculated. Saponins. At Michigan State University, initial experiments will determine if soybean saponins are absorbed by humans. Lack of pure standards and inadequate methodology have previously prevented determination of soy saponin absorption. Both have been overcome by Dr. M.A. Berhow at the USDA, ARS, National Center for Agricultural Utilization Research in Peoria, IL, who will work with Dr. Bennink on the project. Blood and urine samples from men and women consuming saponins will be analyzed for saponins using liquid chromatography B electrospray ionization B mass spectroscopy analysis (80). If saponins are detected in blood and urine, then the ADME of soybean saponins will be determined. Carnosine. Massachusetts researchers will look at the ADME of carnosine, which is an antioxidant derive from beef products. They will also ascertain the effect of this bioactive on lipid oxidation. Omega 3 Fatty Acids. Bioavailability of Omega 3 fatty acids will be investigated at Massachusetts. Studies will focus on functional food products in which Omega 3 fatty acids have been physically entrapped to prevent oxidation. Isoflavones and Antioxidant Vitamins. A number of plant flavonoid compounds affect the activities of monoxygenases (cytochrome P450) involved in Phase 1 metabolism of endogenous and exogenous compounds (2). Soy isolate induces Phase II enzyme metabolism (conjugation reactions) by increasing the activity of hepatic glutathione S-transferase, quinone reductase, and uridine 5'-diphosphate glucuronosyltransferase (81). Because of the importance of B-carotene, lutein, and vitamins A and E to Phase II enzymes, the UC Davis station will characterize the effect of soy isoflavones on the ADME of these key nutrients. Free-living volunteers will consume a control or soy supplemented diet in random crossover design . The low dose (~ 1/10 current DRI values) of radiolabeled (50 nCi) 14C-labeled B-carotene, lutein, and vitamins A, E and folate will be followed in blood, urine, and feces. Labeled intact nutrients and their labeled metabolites will also be measured in plasma and urine to correlate them with phytonutrient levels. The AUC of t1/2a, t1/2d, and t1/2e, and RT of 14C-labeled B-carotene and vitamins A, E and folate will be calculated. Effects on metabolism of the nutrients and possible interactions among them will be evaluated using regression and correlation analyses. Nebraska researchers will make comparisons between phytonutrients and bioactive compounds derived from animal products, particularly bison, on these antioxidant vitamins. Their research will also involve some previously developed carotenoid enhanced chips. Saponins and Antioxidant Vitamins. UC Davis and Michigan State will determine if the ADME of B-carotene and vitamins A and E are affected by saponin consumption. The methodology for B-carotene and vitamins A and E will be as described above. Isoflavones and Calcium. There is a paucity of data in humans on the efficacy and safety of various types, ratios, and doses of isoflavones on preventing bone loss. Based on preliminary data, isoflavones will not affect calcium absorption but will suppress bone resorption. Purdue will use 41Ca, a long-lived isotope, to label the skeleton and monitor perturbations by diet through a collaboration with the Accelerator Mass Spectrometry facility in Core C. Researchers will be able to determine the effect of various types and dosages of isoflavone products sequentially in the same postmenopausal women on suppressing bone resorption by measuring changes in 41Ca excretion in the urine. Ethnicity, Age or Gender and Calcium. Black and white females at various life stages will be studied at Purdue using metabolic balance and stable calcium isotope kinetic analysis to determine calcium and sodium handling and changes in weight and blood pressure on a high salt diet. Racial differences in response to dietary sodium will be related to changes in sodium handling (plasma renin activity, serum aldosterone, angiotensinogen) and calcium-regulating hormones, and biochemical markers of bone turnover. Underlying mechanisms for racial differences in sodium, and consequently, calcium handling unconfounded by dietary influence will be explored. Among the possible inherent racial differences, regulators of sodium handling including plasma angiotension II, urinary angiotensinogen, urinary and plasma aldosterone, and plasma renin activity will be measured. DNA will be collected for genetic typing of variants in sodium transporters associated with hypertension as part of a separate funding request. This will improve the understanding of the greater incidence of hypertension and lower incidence of osteoporosis in blacks compared to whites. New Mexico State researchers will compare the bone status via DXA and biological indices of Mexican women who derive most of their calcium from dairy versus non dairy sources. Special attention will be paid to the impact of age and the consumption of chile, which is a capsaicin containing compound that results in increased loss of calcium via the feces [49]. Hormone/estrogen replacement therapy (H/ERT) versus botanicals in menopausal women will be assessed using 41Ca, a long-lived radioisotope. 41Ca appearance in the urine reflects resorption of primarily trabecular bone. This method provides a direct measure of bone resorption and is model independent. At Purdue, calcium retention in boys will be determined over a range of calcium intakes between 700-2100 mg/d during three week metabolic camps. Stable isotopes of calcium will be administered orally and by intravenous injection after first week equilibrium period in a subset of boys. Complete urine and fecal collections and blood samples will provide data for multicompartmental and stochastic analysis of calcium metabolism. Bone mass and total body and skeletal site calcium measured by DXA, hormone levels (PTH, vitamin D metabolites, IGF-1, IGF-BP3, estrogen, testosterone, and SHBG), and biochemical markers of bone turnover (serum osteocalcin and bone specific alkaline phosphatase and urinary collagen crosslinks) will be determined in all boys. The relationship of calcium retention and bone turnover to parameters of sexual maturity and size will be analyzed. Optimizing calcium accretion during this growth period should maximize peak bone mass within the genetic potential and reduce risk of osteoporosis later. Iron. At Kansas State, Long-Evans rats fed a control diet (AIN), a diet deficient in iron, a diet low in calcium (0.1% ) or one low in both will be evaluated. Bone radiograms of the femur and tibia will be used to determine if osteopenia using an Instron resulted from the treatments. Total bone ash will be determined and the bone strength will be determined using an Instron. These data will help in understanding how iron could affect bone structure and strength, especially when combined with compromised calcium intake. Arizona will evaluate the boundary between iron deficiency and iron sufficiency after erythropoietic stimulus of neonatal rats receiving low to moderate levels of dietary iron. These researchers will also evaluate the boundary between iron sufficiency and iron toxicity after erythropoietic stimulus of neonatal rats receiving moderate to high levels of dietary or parenterally injected iron. Vitamin B6. The vitamin B6 status of men and women will be studied at Washington State University using long-term controlled metabolic conditions. The effects of vitamin B6 and methionine intakes on metabolite profiles and homocysteine responses will be evaluated to provide a basis for the use of plasma homocysteine concentrations as a functional measure of vitamin B6 status. Information will also be obtained on the relationship between vitamin B6 intake and cigarette smoking by studying functional measures of vitamin B6 and folate metabolism which are associated with DNA damage and cancer. Plasma, erythrocyte, lymphocyte and urinary concentrations of vitamin B6 metabolites will be determined. Activities of erythrocyte alanine and aspartate aminotransferase will be assessed with and without added PLP. Urine will be analyzed for 4-pyridoxic acid. Fasting and post-methionine load plasma tHcy will be assessed. Lymphocytes will be analyzed for DNA uracil content, strand breaks, and apurinic/apyrimidinic sites. Lymphocyte serine hydroxymethyltransferase activity will be analyzed in the presence and absence of excess PLP. Oregon State University researchers will collaborate with Washington State University related to the ADME of vitamins B6 and B12 and folate. At Oregon State, research will further focus on these vitamins and homocysteine concentrations and rheumatoid arthritis. This study will also address the effect of exercise on the above noted nutrients and condition. Folate. At UC Berkeley, human genes involved in folate metabolism will be characterized and the effects of polymorphisms in these genes on folate-dependent metabolic cycles and on the dietary requirement for folate assessed. Cell culture models for assessing folate requirements and metabolism will be developed by transfecting cells with human cDNAs for folate-requiring enzymes. Heterozygous and homozygous mouse models for deletion of genes encoding folate-dependent enzymes will be constructed. The effects of genetic heterogeneity in folate genes on folate metabolism and requirements, and one carbon metabolic fluxes, will be analyzed in these model cell culture and animal systems. Other. Colorado State researchers will explore interaction of phytonutrients with other diet factors on apoptosis in cancerous cell lines, with a focus on cultured colon cells. Initial efforts will be directed at detecting dose-response increases in apoptosis in cells exposed to graded levels of phytonutrients such as lycopene, EGCG, resverratrol and sulforaphane. Once models that respond to graded phytonutrients are worked out, interaction of phytonutrients with other diet factors know to affect apoptosis, such as calorie restriction and omega 3 fatty acids will be evaluated. The morphological method of Duke and Cohen (82) will be used to detect apoptotic cell death and additional assays to evaluate the mechanism of apoptosis, such as caspase-3 and -9 activity, PARP inactivation, annexin V binding and mitochondrial cytochrome C release will be used. Objective 2. Collaboratively develop and maintain a website for dissemination of technical and lay publications related to bioavailability A bioavailability web site will be collaboratively developed. The site will have at least the following information: * List of scientists and their respective stations involved with the W-143 project; * Links to relevant web sites with reliable nutrient information, particularly bioavailability information; * A series of collaborative publications on the state of bioavailability knowledge related to key nutrients studied by W-143 scientists; * A series of collaborative nutrition education publications related to key nutrients studied by W-143 scientists; * Techniques for assessing nutrient bioavailability being used by W-143 researchers; and * Links to each of the AES and CES web sites in the United States. All technical and nutrition education materials included on this site will be peer reviewed prior to publication. The site will be maintained by the New Mexico State University (NMSU) researcher. Collaborative technical and lay publications will be solicited by the NMSU webmaster who will send the materials for review and revision prior to posting on the site. W-143 researchers will work with CES faculty at the various stations on lay publications. All materials will be copyrighted. To facilitate printing, materials will be put in .pdf format. To enhance access, letters will be sent to all AES and CES Directors asking that a link to the W-143 site be included on each of the respective web pages associated with each land grant in the United States. Objective 3. Foster collaboration among stations in the development, validation, standardization, and application of technologies, samples, substrates and other resources. Over the long tenure of this project, W-143 researchers, particularly those at the smaller AESs, have been able to do projects that would not otherwise be possible at smaller stations. In addition, researchers at some stations have facilities, such as human metabolic units, that are not available at other stations. During the last 5 year window this led to collaborative efforts such as: * Sharing of samples collected in a human metabolic study which were analyzed for aspects beyond the original scope of the human metabolic study; * Hydroponically grown and isotopically labeled foodstuffs at Purdue which were used at several other stations; * Supplying proprietary supplements developed in industry for a research project at another W-143 station; * Providing the scientist at a small station with a sabbatical at another station to learn techniques in using radioisotopes; and * Organizing and presenting a symposium on the status of bioavailability research at an Experimental Biology meeting. During the proposed research anticipated activities include: * Michigan State University researcher plans to study phytonutrients (saponins and isoflavones) in conjunction with UC Davis. * UC Davis and Nebraska scientists will investigate the vitamin A activity of B-carotene; * Purdue, New Mexico, and UC Davis stations plan to study calcium. * Oregon State, UC Berkeley and Washington State University researchers will collaborate on research related to vitamins B6, B12 and folate. * Michigan, Nebraska, Purdue, New Mexico, and UC Davis stations will develop, validate, standardize, and/or apply some common analytic protocols.

Measurement of Progress and Results

Outputs

• Provide significant contributions to the body of knowledge associated with each of phyto- or essential nutrients studied
• Provide background materials for nutritionists to more accurately estimate the amounts of foods needed for adequate intake of a given nutrient
• Provide the agriculture sector, the food industry and health promotion and disease prevention professionals with data that will potentially result in the following outcomes or impacts

Outcomes or Projected Impacts

• Improving nutrient availability via genetic alteration of livestock and crops
• Altering food processing to improve bioavailability and stability of inherent nutrients
• Development of value-added products that have nutrients associated with decreased risk of disease that better enable the public to apply the Dietary Guidelines for Americans
• Data from this project will be available for Cooperative Extension personnel in each of the states involved. Through the developed website, AES researchers, CES faculty and other non bioavailability researchers will have access to information on techniques used to determine bioavailability. The website will also provide resources for nutrition education of the lay public.
• As in the past, researchers associated with this project will serve on panels and/or provide input related to specific nutrients (e.g., Drs Weaver - Calcium; Dr Clifford on Folate and B- carotene; Dr Shultz-Vitamin B6) for the DRIs/AIs and for the Food and Drug Advisory Committee on Fortification and Food Safety (e.g., Dr Clydesdale).

Milestones

(2002): Development of the baseline aspects of the W-143 Nutrient Bioavailability website will be completed during the first six months of the new project cycle. Researchers will write peer reviewed articles related to research findings. Data will be presented at professional meetings. Researchers will write grants for additional funding of projects at various stations.

(2003): Technical and lay resources will be posted on an ongoing basis. Researchers will write peer reviewed articles related to research findings. Data will be presented at professional meetings. Researchers will write grants for additional funding of projects at various stations.

(2004): Technical and lay resources will be posted on an ongoing basis. Researchers will write peer reviewed articles related to research findings. Data will be presented at professional meetings. Researchers will write grants for additional funding of projects at various stations.

(2005): Technical and lay resources will be posted on an ongoing basis. Researchers will write peer reviewed articles related to research findings. Data will be presented at professional meetings. Researchers will write grants for additional funding of projects at various stations.

(2006): Technical and lay resources will be posted on an ongoing basis. Researchers will write peer reviewed articles related to research findings. Data will be presented at professional meetings. Researchers will write grants for additional funding of projects at various stations.

(0):0

Projected Participation

View Appendix E: Participation

Outreach Plan

The outreach for this project has been addressed in Objective 2. Details are described in the Methods section. In addition, researchers will submit manuscripts for publication in peer reviewed journal and make peer reviewed professional presentations at local, state and national meetings.

Organization/Governance

The Technical Committee will be composed of one voting member from each station. Other station representatives may contribute to the stations research objectives and participate in the Committees annual meeting and Committee activities. The Administrative Advisor and CSREES representative will provide guidance for the Technical Committee. The Committee officers, elected annually, will be: Chairperson, Secretary, and Chairperson Elect

The Chairperson Elect will become the Chairperson the following year. The Chairperson Elect will perform such functions as the committee and/or Chairperson may designate and may chair Technical Committee meetings in the absence of the Chairperson. The office of Chairperson Elect will provide continuity in leadership. The Chairperson and Secretary will perform their customary duties such as preparation of minutes and annual reports..

The Technical Committee will meet annually with the place and date determined by the Committee and approved by the Administrative Advisor. The annual Technical Committee Meeting will rotate among the various member stations by invitation. The location and approximate dates of the next annual meeting will be determined during the previous years meeting. The Chairperson will finalize dates and meeting arrangements with the voting member of the host station after consultation with other members of the Technical Committee.

Literature Cited

(1) Kessler RC, Davis RB, Foster DF, Van Rompay MI, Walters EE, Wilkey SA et al. Long-term trends in the use of complementary and alternative medical therapies in the United States. Annals of Internal Medicine 2001; 135(4):262-268.

(2) Evans AM. Influence of dietary components on the gastrointestinal metabolism and transport of drugs. Therapeutic Drug Monitoring 2000; 22(1):131-136.

(3) Helsby NA, Chipman JK, Gescher A, Kerr D. Inhibition of mouse and human CYP 1A- and 2E1-dependent substrate metabolism by the isoflavonoids genistein and equol. Food and Chemical Toxicology 1998; 36(5):375-382.

(4) Parker RS, Sontag TJ, Swanson JE. Cytochrome P4503A-dependent metabolism of tocopherols and inhibition by sesamin. Biochemical and Biophysical Research Communications 2000; 277(3):531-534.

(5) Mitchell GV, Cook KK, Jenkins MY, Grundel E. Supplementation of rats with a lutein mixture preserved with vitamin E reduces tissue phylloquinone and menaquinone-4. International Journal for Vitamin and Nutrition Research 2001; 71(1):30-35.

(6) Wilhelm C. Growing pains in botanicals and herbal supplements. Chemical Market Reporter 256[19], 12-13. 1999.

(7) Challener C. Specialty products are the bright spot in ditary supplements. Chemical Market Reporter 260[8], 3-10. 2001.

(8) USDA. USDA NRICGP 2002 Program Description and Guidelines for Proposal Preparation. Cooperative State Research, Education and Extension Service, United States Department of Agriculture . 2001. 11-10-2001.

(9) Graves E. National Hospital Discharge Survey: Annual Summary, 1993. Vital Health Statistics 13[121]. 1995.

(10) Ray NF, Chan JK, Thamer M, Melton LJ. Medical expenditures for the treatment of osteoporotic fractures in the United States in 1995: Report from the national osteoporosis foundation. Journal of Bone and Mineral Research 1997; 12(1):24-35.

(11) Pines A, Katchman H, Villa Y, Mijatovic V, Dotan I, Levo Y et al. The effect of various hormonal preparations and calcium supplementation on bone mass in early menopause. Is there a predictive value for the initial bone density and body weight? Journal of Internal Medicine 1999; 246(4):357-361.

(12) Recker RR, Davies KM, Dowd RM, Heaney RP. The effect of low-dose continuous estrogen and progesterone therapy with calcium and vitamin D on bone in elderly women - A randomized, controlled trial. Annals of Internal Medicine 1999; 130(11):897

(13) Knight DL-WP, Eden J. A review of phytoestrogens and their effects in relation to menopausal symptoms. Australian Journal of Nutrition and Dietetics 53, 5-11. 1996.

(14) Kurzer MS, Xu X. Dietary phytoestrogens. Annual Review of Nutrition 1997; 17:353-381.

(15) Eriksen Ef, Colvard Ds, Berg Nj, Graham Ml, Mann Kg, Spelsberg Tc et Al. Evidence of Estrogen-receptors in Normal Human Osteoblast-like Cells. Science 1988; 241(4861):84-86.

(16) Knight D, Eden J. A review of the clinical effects of phytoestrogens. Obstetrics and Gynecology 63, 735. 1996.

(17) Nagata C, Takatsuka N, Kurisu Y, Shimizu H. Decreased serum total cholesterol concentration is associated with high intake of soy products in Japanese men and women. Journal of Nutrition 1998; 128(2):209-213.

(18) Tsuda M, Kitazaki T, Ito T, Fujita T. The effect of ipriflavone (tc-80) on bone-resorption in tissue- culture. Journal of Bone and Mineral Research 1986; 1(2):207-211.

(19) Tsutsumi H. Biological Pharmacological Bulletin 18, 1012-1015. 1995.

(20) Arjmandi BH, Alekel L, Hollis BW, Amin D, StacewiczSapuntzakis M, Guo P et al. Dietary soybean protein prevents bone loss in an ovariectomized rat model of osteoporosis. Journal of Nutrition 1996; 126(1):161-167.

(21) Anderson JJB, Ambrose WW, Garner SC. Biphasic effects of genistein on bone tissue in the ovariectomized, lactating rat model. Proceedings of the Society for Experimental Biology and Medicine 1998; 217(3):345-350.

(22) Draper CR, Edel MJ, Dick IM, Randall AG, Martin GB, Prince RL. Phytoestrogens reduce bone loss and bone resorption in oophorectomized rats. Journal of Nutrition 1997; 127(9):1795-1799.

(23) Stormer Fc, Reistad R, Alexander J. Glycyrrhizic acid in licorice - Evaluation of health-hazard. Food and Chemical Toxicology 1993; 31(4):303-312.

(24) Lasztity R, Hidevegi M, Bata A. Saponins in food. Food Reviews International 1998; 14(4):371-390.

(25) Yoshiki Y, Kudou S, Okubo K. Relationship between chemical structures and biological activities of triterpenoid saponins from soybean. Bioscience Biotechnology and Biochemistry 1998; 62(12):2291-2299.

(26) Vogel V, Newman RA, Ainslie N, Winn R. Phase I pharmacology and toxicity study of glycyrrhetinic acid as a chemoprevenative drug. Proceedings of American Association of Cancer Research 33, 208. 1992.

(27) Adlercreutz H, van der Wildt J, Kinzel J, Attalia H, Wahala K, Makela T et al. Lignan and isoflavonoid conjugates in human urine. Journal of Steroid Biochemistry and Molecular Biology 1995; 52(1):97-103.

(28) Siess MB, Leclerc J, Canivenclavier MC, Rat P, Suschetet M. Heterogenous effects of natural flavonoids on monooxygenase activities in human and rat-liver microsomes. Toxicology and Applied Pharmacology 1995; 130(1):73-78.

(29) Onozawa M, Fukuda K, Ohtani M, Akaza H, SUGIMURA T, Wakabayashi K. Effects of soybean isoflavones on cell growth and apoptosis of the human prostatic cancer cell line LNCaP. Japanese Journal of Clinical Oncology 1998; 28(6):360-363.

(30) Kucuk O, Sarkar F, Sakr W, Djuric Z, Pollak M, Khachik F et al. Phase II randomized clinical trial of lycopene supplementation before radical prostatectomy. Cancer Epidemiology Biomarkers & Prevention 2001; 10:861-868.

(31) Ahmad N, Gupta S, Mukhtar H. Green tea polyphenol epigallocatechin-3-gallate differentially modulates nuclear factor kappa B in cancer cells versus normal cells. Archives of Biochemistry and Biophysics 2000; 376(2):338-346.

(32) Wenzel U, Kuntz S, Brendel M, Danial H. Dietary flavone is a potent apoptosis inducer in human colon carcinoma cells. Cancer 2000; 60:3823-3831.

(33) Bonnesen C, Eggleston I, Hayes JD. Dietary indoles and isothiocynanates that are generated from cruciferous vegetables can both stimulate apoptosis and confer protection against DNA damage in human colon cell lines. Cancer Research 2001; 61:6120-6130.

(34) Gamet-Payrastre L, Li P, Lumeau S, Cassar G, Dupont M, Chevolleau S et al. Sulforaphane, a naturally ocurring isothiocyanate, induces cell cycle arrest and apoptosis in HT29 human colon cancer cells. Cancer Research 2000; 60:1426-1433.

(35) Sumantran VN, Zhang R, Lee DS, Wicha MS. Differential regulation of apoptosis in normal versus transformed mammary epithelium by lutein and retinoic acid. Cancer Epidemiology Biomarkers & Prevention 2000; 9(3):257-263.

(36) Surh YJ, Hurh YJ, Kang JY, Lee E, Kong G, Lee SJ. Resveratrol, an antioxidant present in red wine, induces apoptosis in human promyelocytic leukemia (HL-60) cells. Cancer Letters 1999; 140(1-2):1-10.

(37) Xu K, Thornalley PJ. Studies on the mechanism of the inhibition of human leukaemia cell growth by dietary isothiocyanates and their cysteine adducts in vitro. Biochemical Pharmacology 2000; 60(2):221-231.

(38) Khar A, Ali AM, Pardhasaradhi BVV, Begum Z, Anjum R. Antitumor activity of curcumin is mediated through the induction of apoptosis in AK-5 tumor cells. Febs Letters 1999; 445(1):165-168.

(39) Kelly M, Geigerman C, Loo G. Epigallocatechin gallate protects U937 cells against nitric oxide-induced cell cycle arrest and apoptosis. Journal of Cell Biochemistry 2001; 81:647-658.

(40) Hibasami H, Komiya T, Achiwa Y, Ohnishi K, Kojima T, Nakanishi K et al. Black tea theaflavins induce programmed cell death in cultured human stomach cancer cells. International Journal of Molecular Medicine 1998; 1(4):725-727.

(41) Atten M, Attar B, Milson T, Holian O. Lycopene and 1,25-dihydroxyvitamin D3 cooperate in the inhibition of cell cycle progression and induction of differentiation in HL-60 leukemic cells. Nutritional Biochemistry 2001; 62:1423-1432.

(42) Awad A, Fink C. Phytosterols as anticancer dietary components: Evidence and mechanism of action. Journal of Nutrition 2000; 130:2127-2130.

(43) Ahmad N, Feyes DK, Nieminen AL, Agarwal R, Mukhtar H. Green tea constituent epigallocatechin-3-gallate and induction of apoptosis and cell cycle arrest in human carcinoma cells. Journal of the National Cancer Institute 1997; 89(24):1881-1886.

(44) Perry T, Hansen S, Tischler B, Bunting R, Barry K. A new metabolic disorder associated with neurologic disease and mental defect. New England Journal of Medicine 1967; 277:1219-1227.

(45) United States Department of Agriculture, United States Department of Health and Human Services. Nutrition Monitoring in the United States. [DHHS Publication No. (PHS) 89-1255]. 1989. Hyattsville MD, United States Government Printing Office.

(46) Nickel KP, Martin BR, Smith DL, Smith JB, Miller GD, Weaver CM. Calcium bioavailability from bovine milk and dairy products in premenopausal women using intrinsic and extrinsic labeling techniques. Journal of Nutrition 1996; 126(5):1406-1411.

(47) Heaney RP, Recker RR, Weaver CM. Absorbability of calcium sources - the limited role of solubility. Calcified Tissue International 1990; 46(5):300-304.

(48) Andon MB, Peacock M, Kanerva RL, DeCastro JAS. Calcium absorption from apple and orange juice fortified with calcium citrate malate (CCM). Journal of the American College of Nutrition 1996; 15(3):313-316.

(49) Heaney RP, Weaver CM. OXALATE - EFFECT ON CALCIUM ABSORBABILITY. American Journal of Clinical Nutrition 1989; 50(4):830-832.

(50) Mejia D. Impact of Chile on Selected Milk Constituents from Dairy Cows fed Chile and Impact of Chile on Calcium Parameters in Rats fed Chile in Diets with Calcium Derived from Milk. New Mexico State University, 1995.

(51) Cofactors influencing the calcium requirement-other nutrients.: 1994.

(52) Nordin BEC, Need AG, Morris HA, Horowitz M. The nature and significance of the relationship between urinary sodium and urinary calcium in women. Journal of Nutrition 1993; 123(9):1615-1622.

(53) Shortt C, Madden A, Flynn A, Morrissey PA. Influence of dietary-sodium intake on urinary calcium excretion in selected Irish individuals. European Journal of Clinical Nutrition 1988; 42(7):595-603.

(54) Breslau NA, Mcguire JL, Zerwekh JE, Pak CYC. The role of dietary-sodium on renal excretion and intestinal- absorption of calcium and on vitamin-d metabolism. Journal of Clinical Endocrinology and Metabolism 1982; 55(2):369-373.

(55) Wastney M, Ng D, Smith D, Martin B, Peacock M, Weaver C. Difference in calcium kinetics between adolescent girls and young women. American Journal of Physiology 1996; 271:R208-R216.

(56) Heaney RP, Recker RR, Saville PD. Menopausal changes in calcium balance performance. Journal of Laboratory and Clinical Medicine 1978; 92(6):953-963.

(57) Martin BR, Wastney ME, Ng J, Smith D, Peacock M, Weaver CM. Changes in calcium kinetics with post pubertal age. Journal of Bone and Mineral Research 1997; 12:S534.

(58) Godber JS. Nutrient bioavailability in humans and experimental-animals. Journal of Food Quality 1990; 13(1):21-36.

(59) Shils M, Olson J, Shike M. Modern Nutrition in Health and Disease. 8th ed. Philadelphia, PA: Lea & Febiger, 1994.

(60) Merrill AH, Henderson JM, Wang E, McDonald BW, Millikan WJ. Metabolism of vitamin-b-6 by human-liver. Journal of Nutrition 1984; 114(9):1664-1674.

(61) Miller LT, Kerkvliet NI. Effect of vitamin-b6 on immunocompetence in the elderly. Annals of the New York Academy of Sciences 1990; 587:49-54.

(62) Verhoef P, Stampfer MJ, Buring JE, Gaziano JM, Allen RH, Stabler SP et al. Homocysteine metabolism and risk of myocardial infarction: Relation with vitamins B-6, B-12, and folate. American Journal of Epidemiology 1996; 143(9):845-859.

(63) Rimm EB, Willett WC, Hu FB, Sampson L, Colditz GA, Manson JE et al. Folate and vitamin B-6 from diet and supplements in relation to risk of coronary heart disease among women. Jama-Journal of the American Medical Association 1998; 279(5):359-364.

(64) Dudman N, Wilchen DWJ, Lynch J, Macey D, Lundberg P. Disordered methionine/homocysteine metabolism in premature vascular disease. Journal of Clinical Investigation 1996; 98:177-184.

(65) Ubbink JB, Vandermerwe A, Delport R, Allen RH, Stabler SP, Riezler R et al. The effect of a subnormal vitamin B-6 status on homocysteine metabolism. Journal of Clinical Investigation 1996; 98(1):177-184.

(66) Stabler SP, Sampson DA, Wang LP, Allen RH. Elevations of serum cystathionine and total homocysteine in pyridoxine-, folate-, and cobalamin-deficient rats. Journal of Nutritional Biochemistry 1997; 8(5):279-289.

(67) Martinez M, Cuskelly GJ, Williamson J, Toth JP, Gregory JF. Vitamin B-6 deficiency in rats reduces hepatic serine hydroxymethyltransferase and cystathionine beta-synthase activities and rates of in vivo protein turnover, homocysteine remethylation and transsulfuration. Journal of Nutrition 2000; 130(5):1115-1123.

(68) Reisenauer AM, Halsted CH. Human Folate Requirements. Journal of Nutrition 1987; 117(3):600-602.

(69) Rodriguez M. A conspectus of research on folacin requirements of man. Journal of Nutrition 1978; 108(12):1983-2047.

(70) Krinsky N, Mathewsroth M, Welankiwar S, Sehgal P, Lausen N, Russett M. The metabolism of [c-14] beta-carotene and the presence of other carotenoids in rats and monkeys. Journal of Nutrition 1990; 120(1):81-87.

(71) Scholnik A, Arnold D, Walker W, Schwenke P, Suhrland L, Balcueva E High-dose folinic acid and 5-fluorouracil in the treatment of advanced colon cancer. American Journal of Clinical Oncology-Cancer Clinical Trials 1988; 11(5):558-563.

(72) Bergen HR. Synthesis of deuterated beta-carotene. Methods in Enzymology 1992; 213:49-53.

(73) Dueker SR, Lunetta JM, Jones AD, Clifford AJ. Solid-phase extraction protocol for isolating retinol-d(4) and retinol from plasma for parallel-processing for epidemiologic studies. Clinical Chemistry 1993; 39(11):2318-2322.

(74) Dueker SR, Jones AD, SMITH GM, Clifford AJ. Stable-isotope methods for the study of beta-carotene-d(8) metabolism in humans utilizing tandem mass-spectrometry and high-performance liquid-chromatography. Analytical Chemistry 1994; 66(23):4177-4185.

(75) Novotny JA, Dueker SR, ZECH LA, Clifford AJ. Compartmental analysis of the dynamics of beta-carotene metabolism in an adult volunteer. Journal of Lipid Research 1995; 36(8):1825-1838.

(76) Alpha-Tocopherol B-CCPSG. Alpha-Tocopherol, Beta-Carotene cancer prevention study. New England Journal of Medicine 1994; 330:1129-1135.

(77) Pasatiempo A, Abaza M, Taylor C, Ross A. Effects of timing and dose of vitamin-a on tissue retinol concentrations and antibody-production in the previously vitamin-a-depleted rats. American Journal of Clinical Nutrition 1992; 55(2):443-451.

(78) Folman Y, Russell R, Tang G, Wolf G. rabbits fed on beta-carotene have higher serum levels of all- trans retinoic acid than those receiving no beta-carotene. British Journal of Nutrition 1989; 62(1):195-201.

(79) Lammer E, Chen D, Hoar R, Agnish N, Benke P, Braun J. Retinoic acid embryopathy. New England Journal of Medicine 1985; 313(14):837-841.

(80) Berhow MA, Wagner ED, Vaughn SF, Plewa MJ. Characterization and antimutagenic activity of soybean saponins. Mutation Research-Fundamental and Molecular Mechanisms of Mutagenesis 2000; 448(1):11-22.

(81) Appelt LC, Reicks MM. Soy feeding induces phase II enzymes in rat tissues. Nutrition and Cancer-An International Journal 1997; 28(3):270-275.

(82) Duke R, Cohen J. Morphological and Biochemical Assays of Apoptosis. In: Coligan J, Kruisbeek A, Margulies D, Shevach E, Strober W, editors. Current Protocols in Immunology. New York: John Wiley & Sons, 1992.

Attachments

Land Grant Participating States/Institutions

AZ, CA, CO, CT, IL, IN, KS, MA, ME, MI, NE, OK, OR

Non Land Grant Participating States/Institutions

California-San Diego