Brief Summary of Minutes of Annual Meeting

W-1002 Annual Report 2004 University of California-Davis and University of California-Berkeley: Cellular, metabolic and kinetic effects of genetic polymorphisms on folate metabolism. The Shane laboratory (Berkeley) has continued with studies on folate metabolism of common human genetic polymorphisms that have been shown to influence disease risk. They have developed additional genetic models in mice to mimic the effects of how these polymorphisms influence folate metabolism and development. These new animal models have gene interruptions that disturb mitochondrial folate metabolism and histone methylation, a process involved in regulation of gene expression. They also continue the development of mass spectrometry methods for studying one carbon metabolic fluxes and DNA methylation to allow the study of how nutritional status influences these processes. They have measured one carbon metabolic fluxes, homocysteine remethylation, and DNA methylation in some of the mouse genetic models. Available data indicate that one carbon fluxes are highly influenced by folate and methionine status, and that the influence of genetic variants in folate enzymes on one carbon metabolism is also dependent on nutritional status. They continue to evaluate genetic risk factors for neural tube defects. The genetic and cellular work with the animal models is complemented by the work at the Clifford laboratory (Davis) on kinetic modeling of folate in humans. They have constructed the first kinetic model of the behavior of folate metabolism as it might occur in vivo in humans. They discovered that folate absorption, bile folate flux, and body folate stores are larger than prior estimates. Marrow folate uptake and pteroylpolyglutamate synthesis, recycling, and catabolism are saturable processes. Visceral pteroylpolyglutamate is an immediate precursor of plasma p-aminobenzoylglutamate. The model is a working hypothesis with derived features that are explicitly model-dependent. This model successfully quantified folate metabolism as it might occur in vivo in humans. Further testing is underway. Outcome: In order for information on genetic polymorphisms to be useful in determining disease risk, the changes in folate metabolism at the both the cellular and whole body levels for a given polymorphism must be evaluated. These projects, taken together, provide information on this full spectrum. Impact: The results of these studies, taken together, will provide information on how specific polymorphisms of genes involved in folate metabolism alter nutrient metabolism and will provide the means to apply this information to studies in humans that could suggest appropriate individual dietary changes. Kansas State University and the Western Human Nutrition Research Center at the University of California-Davis: Copper metabolism influences factors involved in oxidative stress. In keeping with studies involving the genetic, cellular and metabolic aspects of nutrient metabolism, these groups are evaluating copper. The Medeiros laboratory (Kansas) continues to research the impact of copper-deficiency upon cardiac hypertrophy. A critical part of this research centers on mitochondrial biogenesis as altered by copper deficiency. They have demonstrated that certain transcription factors, such as mitochondrial transcription factor A, that coordinate gene activation of nuclear and mitochondrial genomes of mitochondrial proteins, are up regulated in copper deficiency. Furthermore, this group has demonstrated that mitochondrial potential is markedly reduced in copper deficient rats. Since this latter observation is an early indication of apoptosis, a cell culture model, murine derived C2C1, was used to determine if copper deficiency can decrease mitochondrial membrane potential and subsequently lead to apoptosis. The cells were made copper-deficient by treatment with the copper chelator tetraethylenepentamine (TEPA). In TEPA treated cells, the activity of Cu, Zn- superoxide dismutase and cytochrome c oxidase decreased dramatically. The protein levels of nuclear encoded subunits of the cytochrome c oxidase decreased, but the mitochondrial encoded subunits remained unchanged. Decreased mitochondrial membrane potential was indicated in TEPA-treated cells, but further investigation of the potential induction of apoptosis by measuring caspase-3 activity, protein concentrations of Bcl-2 and Bax, and DNA fragmentation suggested that apoptosis is not induced in TEPA-treated C2C12 cells. Cells with decreased mitochondrial membrane potential were not destined to apoptosis as a result of copper-deficiency. The results of others showing that markers of apoptosis are increased in hearts from copper-deficient rats could be related more to the heart disease state itself, rather than copper-deficiency per se. If chelation of copper can reduce SOD and cytochrome c oxidase, can high copper intake alter antioxidant status as well? The Turnland laboratory (Davis) has discovered that when long-term copper intake is high (7.8 mg/d), a substantial amount of copper is retained. Some indices of antioxidant status and immune function also change suggesting a possible adverse effect. Outcome: Clearly, factors that influence copper levels can influence oxidative stress at both the cellular and whole body levels. This appears to be the case for both copper deficiency and high copper levels. Future studies of this nutrient should examine this parameter. Impact: The amount of copper used in the intake studies is less than the current UL for copper, suggesting that the current UL is too high. Washington State University, University of Arizona, Oregon State University: Relationship of Smoking to Nutrient Metabolism. The Winzerling laboratory (Arizona) is conducting studies on the impact of smoking at the cellular level using lung cells. Since the lung cell layer represents a single cell surface interface between lung and blood, events in lung tissues can influence whole body antioxidant and nutrient metabolism. Smoke from multiple sources contains iron in the acidic phase as well as numerous compounds that act as oxidants. By the Fenton reaction, if the iron is available, substantive oxidative stress can occur. Normally cells protect against oxidative stress by storing iron in ferritin. However, this group found that exposure of lung cells to low levels of cigarette smoke resulted in a down regulation of ferritin synthesis that was primarily the result of an up regulation in iron regulatory protein 2 (IRP2). IRP2 is one of two proteins primarily responsible for translational control of ferritin synthesis. These results indicate that ferritin levels are reduced at a time when iron storage is desirable and implies that iron-mediated oxidative stress could be, in part, responsible for the oxidative stress observed in smokers. To complement these studies on the impact of smoking on nutrient metabolism and oxidative stress, evaluation of the impact of smoking on whole body human nutrient status by intervention studies are underway. Smokers exhibit reduced B-6 nutritional status and previous research indicates serine hydroxymethyltransferase (SHMT) declines during vitamin B-6 (B-6) deficiency. The Shultz group (Washington) is studying the effects of smoking on SHMT with the consumption of B-6 supplements. They studied smokers during successive 28-d periods. Healthy smokers (S; n=6) and nonsmokers (NS; n=6) consumed controlled diets providing 0.7 (depletion), 1.4 (repletion 1) and 2.2 (repletion 2) mg B-6/d, and a supplemented (10.3 mg B-6/d) self-selected diet. Lymphocyte (LYM) SHMT activity was assayed with (stimulated) and without (basal) added pyridoxal phosphate (PLP). Basal SHMT activity in S was significantly higher (each 35%) than NS after depletion and repletion period 2, respectively. For NS, SHMT activity was significantly higher after supplementation compared to controlled diet periods, and was positively correlated with B-6 intake, plasma (PLA) PLP and 4-pyridoxic (4-PA), LYM PLP, pyridoxamine phosphate and negatively with DNA strand breaks. For S and NS, activity coefficients were inversely correlated with B-6 intake, PLA PLP and 4-PA. These findings indicate that vitamin B-6 intake and smoking exert distinct effects on LYM SHMT activity. In keeping with the effects of smoking on the methyl transfer pathways, the Oregon State group has submitted an NIH grant to determine the effectiveness of an intensive, multi-faceted smoking cessation intervention on abstinence rates in postmenopausal women. Current research shows that older individuals with high blood homocysteine concentrations have more hip fractures than those with lower levels. High homocysteine levels are associated with low intakes of B-vitamins (B-6, folate and B-12) and older individuals are at increase risk of poor intakes of B-vitamins. Thus, there may be protective effects of B-vitamins on fracture risk among current smokers. One objective of this project is to evaluate the consequences of smoking on blood levels of homocysteine and the B-vitamins including Vitamin B-6. Outcome: Smoking can influence metabolism of iron by lung tissues and of vitamin B-6 at the whole body level. These changes could is could have a direct effect on methyl transfer inside cells and ultimately on homcysteine metabolism. Compounds in smoke could prevent synthesis of the primary protein that stores iron in lung cells and thereby increase oxidative stress. Compounds in smoke also influence the use of the vitamin B-6. Impact: Supplements of the B vitamins and consumption of foods high in antioxidants could be required to improve stress response and adverse effects of smoking. The Oregon State group also is recruiting subjects for a study that will examine whether physical activity at the levels recommended by the IOM increases blood homocysteine levels in active individuals compared to inactive controls, while monitoring dietary intakes of B-vitamins and blood status measures. The results of this study could suggest whether exercise should be considered as a variable in future studies of B-vitamin status in smokers. Purdue University, Oregon State University, Kansas State University: Physical Activity, Calcium, Iron and Vitamin Intake Impact on Bone Formation and Bone Health. A central hypothesis of the study described above at Oregon State is that an intensive, multi-faceted intervention will decrease smoking behavior and improve bone health in post-menopausal women. The Oregon group will evaluate the effects of the cessation of smoking on longitudinal changes in bone health and intakes of bone building nutrients in the diet. They also will monitor changes in physical activity for impact on bone health in this population. This study will contribute to the collaborative efforts of work by the W-1002 group on bone formation and bone health. In this area this year, two major studies were by the Medeiros laboratory (Kansas) in animals. One study was conducted on outright iron-deficiency and calcium deficiency with a pair-fed group to correct for the decreased food consumed by rats in the iron-deficient groups. The hypothesis of whether iron-deficiency had direct adverse impacts on vertebral trabecular bone and long bones was answered by this study. Four groups of female weanling rats were fed for five weeks diets that were 1) control; 2) calcium restricted, 1.0 g Ca/kg diet; 3) iron deficient, less than 8 mg Fe/kg diet; or 4) control pair-fed to the iron-deficient group. Whole body and femur DEXA analysis revealed that calcium-restricted and iron-deficient rats had lower bone mineral density (BMD) and content (BMC) than pair-fed and controls. However, pair-fed rats also had decreased BMD and BMC compared to controls. The third lumbar trabecular bone microarchitecture in both diet restricted groups had decreased bone volume fraction (BV/TV) and trabecular number and thickness, a less favorable structural model index, and increased trabecular separation compared with the controls and the pair-fed groups as determined by microcomputer tomography. The control or pair-fed groups did not differ from one another, suggesting that iron-deficiency and calcium-restriction had impacts on vertebrae independent of food intake and body weight. Finite element analysis revealed decreased force to compress the vertebrae, decreased stiffness but greater von Mises stress in calcium-restricted and iron-deficient groups compared to controls and pair-fed groups. Urinary deoxypyridinium crosslinks, serum osteocalcin and cholcalciferol were increased in calcium-restricted rats compared to the other 3 groups. Using Micro-CT imaging technology, this study demonstrated micro-architectural pathology due to iron deficiency upon vertebral trabecular bone, compared to controls and pair-fed rats, though not to the same extent as severe-calcium restriction. Since the above study demonstrated compromise in bone in outright deficiency, the Medeiros laboratory then asked whether marginal intakes of iron and/or calcium, likely to be consumed in Western diets, would have any negative impact. Thirty-two female, weanling rats were randomly divided into four groups for 10 weeks: 1) control, 2) calcium restricted (Ca-), 3) iron restricted (Fe-), and 4) calcium and iron restricted (Ca-Fe-). DEXA analysis revealed that marginal Ca- affected bone mineral density (BMD), but marginal Fe- affected whole body bone mineral content (BMC). All treatment groups had decreased BMC and BMD in femurs and the 4th lumbar (L-4) vertebrae. The microarchitecture of the L-4 vertebrae was compromised in Ca-, Fe-, and Ca-Fe- restricted groups: the amount of tissue present in L-4 vertebrae (BV/TV) was reduced in Ca- and Fe- groups; all three treatment groups had decreased trabecular number compared to the control; and both Fe- and Ca-Fe- groups had greater trabecular separation compared to the iron adequate groups. Finite element analysis revealed that L-4 vertebrae from the Fe- group would require less total force to break than Fe adequate groups and the combined Ca-Fe- group. All three treatment groups experienced greater internal stress (Von Mises stress) when force was applied than the control group. Marginal iron restriction, such as that which occurs among some human populations, does reduce bone strength and microarchitecture. The findings of the Medeiros group in animals were extended by the work of the Weaver laboratory (Purdue) on the effects of phytonutrients on calcium metabolism and bone. They conducted a study on the effect of kudzu isoflavones on bone health in 6 month OVX Sprague Dawley rats. Kudzu root powder providing 0.9% isoflavones in the diet increased bone mineral density at several bone sites using pQCT, but had estrogenic effects on the uterus. This Weaver group extended the isoflavone work in a dose response study of the effect of soy isoflavones in a soy protein powder on bone loss in postmenopausal women using a novel 41Ca technology. Thirteen postmenopausal women (> 6 years since menopause) were pre-dosed with 41Ca intravenously. After a 100-day baseline period, subjects were given 40 grams of soy protein per day that contained 0, 97.5, or 135.5 mg of total isoflavones in randomized order. The soy protein isolate powder was incorporated into baked products and beverages. Each 50-day treatment phase was followed by a 50-day recovery phase. Serum isoflavone levels and biochemical markers were measured at the end of each phase. Twenty-four hour urine collections were performed every ~10 days during each phase for 41Ca/Ca analysis by Accelerator Mass Spectrometry. Serum isoflavone levels reflected the amount of isoflavones consumed in a dose-dependent manner. None of the isoflavone levels had a significant effect on biochemical markers of bone turnover, urinary cross-linked N-teleopeptides of type I collagen (NTx) and serum osteocalcin (OC), or bone turnover as assessed by urinary 41Ca/Ca. In conclusion, soy isoflavones up to 135.5 mg per day did not suppress bone resorption in postmenopausal women. The Weaver laboratory also evaluated potential racial differences in calcium retention as a function of intake in adolescent girls. They pooled data from five metabolic studies (Camp Calcium) on adolescent black and white girls aged 12-14. Girls were studied during controlled feeding periods of three weeks on diets ranging in calcium intake from 800 to 2300 mg/day. Calcium retention at all intakes was higher in black than white girls. Black women have higher bone mass than white women. This group previously reported that black girls used calcium more efficiently than white girls on one calcium intake. They determined racial differences in the relationship between calcium intake and calcium retention in 82 black and 160 white girls. Black girls utilize calcium more efficiently at all calcium intakes than white girls. University of Maine, University of Nebraska, Purdue University and University of Connecticut: Availability and absorption of nutrients and phytonutrients from food sources. Several studies are designed to determine nutrient availability from food sources or to evaluate nutrient status in a specific population group were completed this year. These studies complement the areas of common research. Accompanying the work in bone health and formation described above, the Weaver laboratory (Purdue) evaluated calcium availability from soy milk. Calcium (Ca) fortified soymilk has gained popularity in the United States. Tricalcium phosphate (TCP) fortified soymilk was shown to have a lower Ca bioavailability than that of cows milk in men. However, the most popular soymilk in the U.S. is fortified with calcium carbonate. The objective of this study was to compare Ca bioavailability of Ca carbonate fortified soymilk and TCP fortified soymilk with cows milk in young healthy women using the dual stable isotope technique. In a 3-way crossover design, 19 volunteers (23+1.9 years old) consumed either cows milk, Ca carbonate fortified soymilk, or TCP fortified soymilk labeled with stable isotopes. Fractional Ca absorption was determined from stable isotope ratios in urine samples. Fractional Ca absorption in Ca carbonate fortified soymilk (0.211+0.057) was not significantly different from that of cows milk (0.198+0.037), but was significantly higher than that of TCP fortified soymilk (0.181+0.039). Soymilk fortified with calcium carbonate is comparable to cows milk, but soymilk fortified with TCP is significantly less well absorbed. Complementing the work in oxidative stress, the Camaire laboratory (Maine) developed and evaluated four beverage formulations. Although the beverages had high anthocyanin levels and antioxidant activity, consumers only found one formulation to be acceptable. This project also has a practical component in collaboration with the Maine Business School conducting an on-line survey to learn more about consumer attitudes towards healthy beverages. The Camaire group will extend this research component next year with a new project to evaluate the literature and recommend a strategy for a qualified health claim for wild blueberries. As part of the project a database of relevant research articles has been compiled. Data from the blueberry supplement and cognition study are being analyzed. Projects starting this fall include testing the efficacy of cranberry supplements against Helicobacter pylori, and evaluation of the hypoglycemic effects of blueberry flavonoids. Impact: Food consumption is preferred to supplements to provide nutrients and phytonutrients. The study of bioavailability of such from fortified foods as well as the effect of cooking on these constituents is required to utilize the information obtained from cellular, metabolic, kinetic and whole body metabolism studies. Extending the work in phytonutrients, the Connecticut group studied a potential relationship between the plasma cholesterol (CH) and carotenoid (CAR) following egg consumption and whether gender influences plasma carotenoids. Using a cross over design, 40 men and 51 pre-menopausal women were randomly assigned to an egg (640 mg/d additional CH and 600 mcg of lutein/zeaxanthin) or a placebo diet (no additional CH or lutein). Based on response to CH, we analyzed plasma CAR concentrations in a subset of 40 subjects (20 hypo- and 20 hyper-responders). Compared to men, women had an average of 30 and 60% higher concentrations of lutein and zeaxanthin respectively (P < 0.01) during the egg period. In addition, with egg intake, plasma lutein concentration of female hyper-responders was greater (1.16 µmol/L) than hypo-responders (0.89 µmol/L) (P < 0.01). A similar, response was observed in men after egg intake with plasma levels of 0.89 µmol/L and 0.63 µmol/L for hyper-responders and hypo-responders respectively (P<0.01). These results suggest that responses to dietary CH and CAR are related and that gender influences plasma CAR concentrations. Impact: This study has addressed some important factors influencing absorption of carotenoids. This is potentially important for human health because of the role of xanthophylls (specifically lutein and zeaxanthin) in preventing age-related macular degeneration. In addition to the work in carotenoids in adults, the Driskell (Nebraska) laboratory studied intakes of carotenoids in children. They evaluated intake and status of carotenoids, as well as vitamin B-6 and vitamin E for 35 children (19 boys, 16 girls), aged 4-8 years, of Latino immigrants were determined. Data were similar by gender. All subjects had adequate vitamin B-6 status. Plasma alpha-tocopherol concentrations <12 micromoles/Liter were observed in 66% of subjects and <7 micromoles/Liter, in 29%; thus, many subjects were inadequate in vitamin E. The carotenoid intakes and plasma concentrations varied greatly between subjects. In a similar practical research area, the Driskell laboratory evaluated microwave steaming as a cooking method on the yields and beta-carotene retentions for broccoli, carrots, green beans, and sweet potatoes. The yields and nutrient retentions were generally higher when cooked by induction-boiling than microwave-steaming. Trained panelists generally scored the flavor and texture of these vegetables as being significantly different by cooking method, but not the color and overall acceptability. Impact: Most children need to consume more alpha-tocopherol, the active form of vitamin E. Cooking vegetables by induction-boiling appears to be a desirable method in terms of cooking yield, carotenoid retention, and sensory characteristics.