Life Sciences Commons^™

The Rna Cargo In Small Extracellular Vesicles From Chicken Eggs Is Bioactive In C57bl/6j Mice And Human Peripheral Blood Mononuclear Cells Ex Vivo, Deborah Fratantonio, Javaria Munir, Jiang Shu, Katherine Howard, Scott R. Baier, Juan Cui, Janos Zempleni

Department of Nutrition and Health Sciences: Faculty Publications

Small extracellular vesicles (sEVs) and their RNA cargo in milk are bioavailable in humans, pigs, and mice, and their dietary depletion and supplementation elicits phenotypes. Little is known about the content and biological activity of sEVs in foods of animal origin other than milk. Here we tested the hypothesis that sEVs in chicken eggs (Gallus gallus) facilitate the transfer of RNA cargo from an avian species to humans and mice, and their dietary depletion elicits phenotypes. sEVs were purified from raw egg yolk by ultracentrifugation and authenticated by transmission electron microscopy, nano-tracking device, and immunoblots. The miRNA profile …

Potential Interactions Between Diatoms And Bacteria Are Shaped By Trace Element Gradients In The Southern Ocean, Alexa R. Sterling, Laura Z. Holland, Randelle M. Bundy, Shannon M. Burns, Kristen N. Buck, P. Dreux Chappell, Bethany D. Jenkins

OES Faculty Publications

The growth of diatoms in the Southern Ocean, especially the region surrounding the West Antarctic Peninsula, is frequently constrained by low dissolved iron and other trace metal concentrations. This challenge may be overcome by mutualisms between diatoms and co-occurring associated bacteria, in which diatoms produce organic carbon as a substrate for bacterial growth, and bacteria produce siderophores, metal-binding ligands that can supply diatoms with metals upon uptake as well as other useful secondary compounds for diatom growth like vitamins. To examine the relationships between diatoms and bacteria in the plankton (diatom) size class (> 3 mu m), we sampled both …

Preliminary Evidence That Lectins In Infant Soy Formula Apparently Bind Bovine Milk Exosomes And Prevent Their Absorption In Healthy Adults, Ezra Mutai, Alice Kah Hui Ngu, Janos Zempleni

Department of Nutrition and Health Sciences: Faculty Publications

Background: Milk exosomes and their microRNA (miR) cargos are bioavailable. The content of exosomes and miRs is negligible in infant formulas compared to human milk, and dietary depletion of exosomes led to changes in bacterial communities and impaired gut health in juvenile mice. Adverse effects of formula feeding may be compounded by using soy formulas due to exosome binding by abundant lectins in that matrix. The purpose of this study was to assess the bioavailability of milk exosomes and their miR cargos added to soy formula in adults, as well as the potential role of soy lectins in exosome …

The Seasonal Flux And Fate Of Dissolved Organic Carbon Through Bacterioplankton In The Western North Atlantic, Nicholas Baetge, Michael J. Behrenfeld, James Fox, Kimberly H. Halsey, Kristina D.A. Mojica, Anai Novoa, Brandon M. Stephens, Craig A. Carlson

Faculty Publications

The oceans teem with heterotrophic bacterioplankton that play an appreciable role in the uptake of dissolved organic carbon (DOC) derived from phytoplankton net primary production (NPP). As such, bacterioplankton carbon demand (BCD), or gross heterotrophic production, represents a major carbon pathway that influences the seasonal accumulation of DOC in the surface ocean and, subsequently, the potential vertical or horizontal export of seasonally accumulated DOC. Here, we examine the contributions of bacterioplankton and DOM to ecological and biogeochemical carbon flow pathways, including those of the microbial loop and the biological carbon pump, in the Western North Atlantic Ocean (∼39–54°N along ∼40°W) …

Multi-Response Surface Optimisation Of Extrusion Cooking To Increase Soluble Dietary Fibre And Polyphenols In Lupin Seed Coat, Liezhou Zhong, Zhongxiang Fang, Mark L. Wahlqvist, Jonathan M. Hodgson, Stuart K. Johnson

Research outputs 2014 to 2021

The seed coat of the legume lupin which is rich in insoluble dietary fibre is a major by-product in human food applications. Extrusion cooking has been demonstrated to increase desirable soluble dietary fibre in the Australian sweet lupin seed coat. In this study, processing condition of twin-screw extrusion cooking was optimised using a central composite rotatable design to increase soluble dietary fibre in lupin seed coat from 44.17 g/kg up to 113.69 g/kg dry basis. The high levels of polyphenols in the seed coat were retained. The optimal extrusion conditions which achieved maximum levels of soluble dietary fibre, total free …

Pharmacokinetics And Antiangiogenic Studies Of Potassium Koetjapate In Rats, Seyedeh F. Jafari, Fouad Saleih R. Al-Suede, Ashwaq H.S. Yehya, Mohamed B.K. Ahamed, Armaghan Shafaei, Muhammad Asif, Yasser M. Tabana, Amin M.S.A. Majid, Hussein M. Baharetha

Research outputs 2014 to 2021

© 2020 Purpose: Koetjapic acid is an active compound of a traditional medicinal plant, Sandoricum koetjape. Although koetjapic acid has a promising anticancer potential, yet it is highly insoluble in aqueous solutions. To increase aqueous solubility of koetjapic acid, we have previously reported a chemical modification of koetjapic acid to potassium koetjapate (KKA). However, pharmacokinetics of KKA has not been studied. In this study, pharmacokinetics and antiangiogenic efficacy of KKA are investigated. Methods: Pharmacokinetics of KKA was studied after intravenous and oral administration in SD rats using HPLC. Anti-angiogenic efficacy of KKA was investigated in rat aorta, human endothelial cells …

Dataset For: Ecotoxicological Benthic Impacts Of Experimental Oil-Contaminated Marine Snow Deposition, Justine S. Van Eenennaam, Melissa Rohal, Jagos R. Radovic, Thomas B. Oldenburg, Isabel Romero, Albertinka J. Murk, Edwin M. Foekema

C-IMAGE data

This dataset contains experimental data on the effect of artificial marine snow on marine benthic invertebrates, water quality measurements, water nutrient measurements, oxygen measurements, and data on oil biodegradation (n-alkanes). Excel sheet with raw data from the experiment: exposure conditions, biota response, chemistry, and observations are included. This dataset supports the publication: van Eenennaam, J.S., Rohal, M., Montagna, P.A., Radović, J.R., Oldenburg, T.B., Romero, I.C., Murk, A.J. and Foekema, E.M. (2019). Ecotoxicological benthic impacts of experimental oil-contaminated marine snow deposition. Marine Pollution Bulletin, 141, 164-175. doi: 10.1016/j.marpolbul.2019.02.025

Dietary Polyphenols And Their Perceived Health Benefits, Stephanie Russell

Honors Theses

This paper will consist of a compilation of information on polyphenol classes, dietary sources, and potential health benefits. Polyphenols can be classified as flavonoids, phenolic acids, lignans, or stilbenes. These molecules consist of over 500 plant metabolites that come from numerous food sources including various fruits, vegetables, grains, and beverages such as coffee, tea, and wine. The quantity of polyphenols in food vary greatly, as does their bioavailability for use in the body after consumption. Due to antioxidant and anti-inflammatory properties, polyphenols are being studied for effects on reducing the risk of cardiovascular disease. This paper will focus on structural …

Micrornas In Bovine Milk Exosomes Are Bioavailable In Humans But Do Not Elicit A Robust Pro-Inflammatory Cytokine Response, Ezra Mutai, Amanda E. Ramer-Tait, Janos Zempleni

Department of Nutrition and Health Sciences: Faculty Publications

Background: Bovine milk exosomes are studied for their roles as bioactive food compounds and as vehicles for drug delivery. Both lines of investigation converge on immune function, e.g., immune regulation by absorption of microRNAs encapsulated in milk exosomes across species boundaries, and the possibility of exosomes and their cargos triggering an immune response if used in drug delivery. This study assessed the bioavailability of immune-related microRNAs from bovine milk and changes in plasma cytokine concentrations after milk consumption in humans, and the secretion of cytokines by human peripheral blood mononuclear cells (PBMCs) cultured with milk exosomes transfected with immune-relevant microRNAs. …

In Vitro Bioaccessibility Of Novel Low-Crystallinity Phytosterol Nanoparticles In Non-Fat And Regular-Fat Foods, Ali Ubeyitogullari, Ozan Ciftci

Department of Food Science and Technology: Faculty Publications

Crystalline structure of phytosterols leads to poor bioavailability and makes their incorporation into foods challenging. Bioaccessibility of first-of-their-kind low-crystallinity phytosterol nanoparticles impregnated in nanoporous starch aerogels (PS-NSA) was evaluated in non-, low-, and regular-fat solid and aqueous food formulations, namely, granola bars and puddings for the first time. Bioaccessibility of the phytosterol nanoparticles was significantly higher than that of crude phytosterols in all food formulations (p < .05); it was 88.2 and 91.8% for low- or regular-fat granola bars, respectively, whereas bioaccessibility of crude phytosterols was ca. 30% in those formulations. However, decreasing the lipid content to zero resulted in lower phytosterols' bioaccessibilities from both PS-NSA (53%) and crude phytosterols (ca. 16%) in non-fat granola bars. Bioaccessibility of crude phytosterols (2%) was significantly enhanced with PS-NSA (19%) in the pudding formulation. PS-NSA allows preparation of low- and non-fat foods enriched with phytosterols while enhancing the health benefits of phytosterols with smaller doses.

Increasing Cis-Lycopene Content Of The Oleoresin From Tomato Processing Byproducts Using Supercritical Carbon Dioxide And Assessment Of Its Bioaccessibility, Lisbeth Vallecilla Yepez

Department of Food Science and Technology: Dissertations, Theses, and Student Research

In recent years, health- and wellness-promoting foods have been one of the major focus of consumers; therefore, the food industry is increasing its efforts in developing these food products. Tomatoes, tomato-food products and their byproducts have gained special attention in virtue of the carotenoids-rich source that they represent, especially lycopene (~90%). The role of lycopene in human diet has been highlighted as a result of its direct relationship with the decrease of chronic diseases. Nevertheless, lycopene is water-insoluble making its extraction simple with organic-toxic solvents; in addition, lycopene degrades easily with light, time, and storage. Lycopene exist in nature mostly …

A Pes Study Of Factors Influencing Metal Partitioning In Aquatic Systems: “Design Of Experiment As, Cd, Co, Cr, Cu, Pb, Ni, And Zn”, Eid A. Alkhatib, John Rapaglia, Leon Theim

Chemistry & Physics Faculty Publications

Mobility and bioavailability of heavy metals are related to their partitioning amongst suspended sediment and water. A Particle Entrainment Simulator (PES) is used to simulate sediment resuspention in natural surface water systems. The simulations were carried out under various conditions of water/suspended solids conditions. Five factors, each at various levels, are tested collectively: the pH of water at two levels (4 and 8), shear stress on bottom sediment at three levels (0.1, 0.3, and 0.5 N/m2 ) salinity of water at two levels (0.01 and 14.0 ppt), organic matter in sediment at three levels (0.50, 1.93, and 3.80%) and temperature …

Immobilization And Phytotoxicity Reduction Of Heavy Metals In Serpentine Soil Using Biochar, I. Herath, P. Kumarathilaka, A. Navarantne, N. Rajakaruna, M. Vithanage

Biological Sciences

Purpose

Serpentine soils derived from ultramafic rocks release elevated concentrations of toxic heavy metals into the environment. Hence, crop plants cultivated in or adjacent to serpentine soil may experience reduced growth due to phytotoxicity as well as accumulate toxic heavy metals in edible tissues. We investigated the potential of biochar (BC), a waste byproduct of bioenergy industry in Sri Lanka, as a soil amendment to immobilize Ni, Cr, and Mn in serpentine soil and minimize their phytotoxicity.

Materials and methods

The BC used in this study was a waste byproduct obtained from a Dendro bioenergy industry in Sri Lanka. This …

Use Of Esi-Fticr-Ms To Characterize Dissolved Organic Matter In Headwater Streams Draining Forest-Dominated And Pasture-Dominated Watersheds, Yuehan Lu, Xiaping Li, Rajaa Mesfioui, James E. Bauer, R. M. Chambers, Elizabeth A. Canuel, Patrick G. Hatcher

Chemistry & Biochemistry Faculty Publications

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) has proven to be a powerful technique revealing complexity and diversity of natural DOM molecules, but its application to DOM analysis in grazing-impacted agricultural systems remains scarce. In the present study, we presented a case study of using ESI-FTICR-MS in analyzing DOM from four headwater streams draining forest-or pasture-dominated watersheds in Virginia, USA. In all samples, most formulas were CHO compounds (71.8-87.9%), with other molecular series (CHOS, CHON, CHONS, and CHOP (N, S)) accounting for only minor fractions. All samples were dominated by molecules falling in the lignin-like region (H/C …

Concentrations And Sources Of Polycyclic Aromatic Hydrocarbons In Surface Coastal Sediments Of The Northern Gulf Of Mexico, Zucheng Wang, Zhanfei Liu, Kehui Xu, Lawrence M. Mayer, Zulin Zhang, Alexander S. Kolker, Wei Wu

Faculty Publications

Background: Coastal sediments in the northern Gulf of Mexico have a high potential of being contaminated by petroleum hydrocarbons, such as polycyclic aromatic hydrocarbons (PAHs), due to extensive petroleum exploration and transportation activities. In this study we evaluated the spatial distribution and contamination sources of PAHs, as well as the bioavailable fraction in the bulk PAH pool, in surface marsh and shelf sediments (top 5 cm) of the northern Gulf of Mexico. Results: PAH concentrations in this region ranged from 100 to 856 ng g(-1), with the highest concentrations in Mississippi River mouth sediments followed by marsh sediments and then …

Release Of Dissolved Organic Carbon From Seagrass Wrack And Its Implications For Trophic Connectivity, Paul S. Lavery, Kathryn M. Mcmahon, Julia Weyers, Mary C. Boyce, Carolyn Elizabeth Oldham

Research outputs 2013

ABSTRACT: The export of old leaves and stems (wrack) from seagrass meadows provides a mechanism for trophic connectivity among coastal ecosystems. As little of this wrack is consumed by mesograzers, leached dissolved organic carbon (DOC) may determine the importance of wrack as a trophic subsidy. However, few studies have examined the effect of seagrass type or age on the release of DOC or its bioavailability. We examined the amount and composition of DOC released from different wrack: Posidonia sinuosa, Amphibolis antarctica and the alga Laurencia sp. We then examined the effect of age on DOC leaching from P. sinuosa wrack. …

The Antiinflammatory Action And Pharmacokinetics Of A Novel Glucosamine-Based Di-Peptide Aminosugar, Mohammad H. Gilzad-Kohan, Kamaljit Kaur, Fakhreddin Jamali

Pharmacy Faculty Articles and Research

Purpose. We have previously shown favorable in vitro gut permeability for three novel dipeptide esters of glucosamine (GlcN) likely facilitated by the peptide transporter 1 (PepT1). Herein, we report the development of a novel assay for the determination of bioavailability of the peptide ester of interest, the anti-inflammatory properties of a glycine-valine ester derivative of GlcN (GVG) as well as its pharmacokinetics under healthy and inflammatory conditions.

Methods. A pre-column derivatization (with 9-fluorenylmethoxycarbonyl) HPLC assay was developed to study bioavailability of GVG, GlcN or cleaved GlcN in the rats that were cannulated in their right jugular vein for …

Plant Calcium Content: Ready To Remodel, Jian Yang, Tracy Punshon, Mary Lou Guerinot, Kendal D. Hirschi

Dartmouth Scholarship

By identifying the relationship between calcium location in the plant cell and nutrient bioavailability, the plant characteristics leading to maximal calcium absorption by humans can be identified. Knowledge of plant cellular and molecular targets controlling calcium location in plants is emerging. These insights should allow for better strategies for increasing the nutritional content of foods. In particular, the use of preparation-free elemental imaging technologies such as synchrotron X-ray fluorescence (SXRF) microscopy in plant biology may allow researchers to understand the relationship between subcellular location and nutrient bioavailability. These approaches may lead to better strategies for altering the location of calcium …

Bioavailability Of Chlorantraniliprole And Indoxacarb To Eastern Subterranean Termites (Isoptera: Rhinotermitidae) In Various Soils, Neil A. Spomer, Shripat T. Kamble, Blair D. Siegfried

Department of Entomology: Faculty Publications

A laboratory study was conducted to determine the toxicity of indoxacarb and chlorantraniliprole to Eastern subterranean termites, Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae) resulting from topical applications and exposure to treated soil. Soils with varying organic matter (0.57-3.64%) and chemical characteristics were used in termiticide bioassays. Lethal dose resulting from topical application indicated that chlorantraniliprole was two- to 11-foldmoretoxic than indoxacarb. Lethal concentration assays yielded opposite results where concentrations of indoxacarb in soil that caused either 50 or 90% mortality of R. flavipes workers at 48 and 144 h were two- to six-fold lower than chlorantraniliprole. The bioavailability of indoxacarb and …

Effects, Uptake, And Fate Of 2,4,6-Trinitrotoluene Aged In Soil In Plants And Worms, Elly Ph Best, Henry E. Tatem, Kaaren N. Geter, Melissa L. Wells, Brian K. Lane

US Army Corps of Engineers

The present study was aimed at providing data to be used at predicting exposure-based effects of 2,4,6-trinitrotoluene (TNT) aged in soil on endpoint organisms representing two trophic levels. These data can be used to define criteria or reference values for environmental management and conducting specific risk assessment. Long-term exposure tests were conducted to evaluate sublethal toxicity and uptake of aged soil-based explosives, with TNT as the main contaminant. In these tests, plants were exposed for 55 d, and biomass and explosives residues were determined. Worms were exposed for 28 and 42 d, and biomass, number, and tissue residues were determined. …

The Key Importance Of Soy Isoflavone Bioavailability To Understanding Health Benefits, T. A. Larkin, William E. Price, L. Astheimer

Faculty of Science - Papers (Archive)

Research over the past two decades has provided significant epidemiological and other evidence for the health benefits of the consumption of soy-based foods. A large number of dietary intervention studies have examined the effects of soy isoflavones on risk factors for cardiovascular disease and hormone-dependent cancers. However, these report large variability in outcome measures, very limited reproducibility between studies and in some cases, controversy between results of clinical trials using dietary soy or soy protein and isoflavone supplementation. This highlights a major gap in our understanding of soy isoflavone uptake, metabolism, distribution, and overall bioavailability. There are many potential factors …

Naturally Present Fatty Acids As Internal Calibrants For Fourier Transform Mass Spectra Of Dissolved Organic Matter, Rachel L. Sleighter, Georgina A. Mckee, Zhanfei Liu, Patrick G. Hatcher

Chemistry & Biochemistry Faculty Publications

The analysis of dissolved organic matter ( DOM) by Fourier transform ion cyclotron resonance mass spectrometry ( FTICR- MS) has gained wide interest recently, driven primarily by its ultrahigh resolving power and mass accuracy. Accurate calibration of mass spectra is a key step to successfully decipher the DOM components. We propose a simple and accurate method to internally calibrate the peaks in the complex spectra without the need to add a calibrant. Mass spectra of DOM samples from the Dismal Swamp, Virginia, and the lower Chesapeake Bay display the presence of naturally occurring fatty acids which can be readily recognized …

Increased Probiotic Yogurt Or Resistant Starch Intake Does Not Affect Isoflavone Bioavailability In Subjects Consuming A High Soy Diet, Theresa A. Larkin, William E. Price, Lee B. Astheimer

Faculty of Science - Papers (Archive)

Objective: Probiotics and prebiotics that affect gut microflora balance and its associated enzymeactivity may contribute to interindividual variation in isoflavone absorption after soy intake, possiblyenhancing isoflavone bioavailability. This study examined the effects of the consumption ofbioactive yogurt (a probiotic) or resistant starch (a known prebiotic) in combination with high soyintake on soy isoflavone bioavailability.Methods: Using a crossover design, chronic soy consumption was compared with soy plusprobiotic yogurt or resistant starch in older male and postmenopausal females (n 31). Isoflavonebioavailability was assessed at the beginning and end of each 5-wk dietary period by samplingplasma and urine after a standardized soy meal.Results: …

Application Of Surrogate Methods For Assessing The Bioavailability Of Pahs In Sediments To A Sediment Ingesting Bivalve, Stuart L. Simpson, Victoria L. Burston, Dianne F. Jolley, Kim Chau

Faculty of Science - Papers (Archive)

The usefulness of two surrogate methods for rapidly determining the bioavailability of PAHs in hydrocarbon-contaminated marine sediments was assessed. Comparisons are made between the PAHs accumulated by the benthic bivalve, Tellina deltoidalis, and the extractable-PAHs determined using a 6-h XAD-2 resin desorption method and a 4-h gut fluid mimic (GFM) extraction method. There were significant positive relationships between PAH bioaccumulation by the bivalves and sediment PAH concentrations. These relationships were not improved by normalising the sediment PAH concentrations to the organic carbon concentration. The average percentage lipid content of the bivalves was 1.47 ± 0.22% and BSAFs for total-PAHs ranged …

Bioavailability Of Uranium And Nickel To Vegetation In A Contaminated Riparian Ecosystem, Tracy Punshon, Karen F. Gaines, Paul M. Bertsch, Joanna Burger

Faculty Research & Creative Activity

The lower portion of Tims Branch (TB), a second-order stream system on the Savannah River site (SC, USA), receives influx of mixed waste-contaminated sediments from Steed Pond, a former settling basin for target processing wastes for over three decades. The magnitude and distribution of U, Ni, and other metals and the potential for trophic movement were studied to facilitate risk assessment and determine potential remedial action. Total and sequential extraction of TB soils demonstrated contaminant heterogeneity both spatially and between operationally defined fractions. Metal concentrations were elevated within riparian zone soils in contrast to stream sediments, suggesting off-site transport. Leaf …

Toxicity And Toxicokinetics Of Cadmium In Capitella Sp. I: Relative Importance Of Water And Sediment As Routes Of Cadmium Uptake, Henriette Selck, Valery E. Forbes, Thomas L. Forbes

Valery Forbes Publications

The importance of dissolved versus sediment-bound cadmium as uptake routes for the deposit-feeding polychaete Capitella species I and the toxicity and toxicokinetics of cadmium from these exposure routes were investigated. Effects were reported as changes in worm growth rate, egestion rate and allometry. Radioactive cadmium (¹⁰⁹Cd) was used as a tracer to examine the uptake (5 d) and subsequent depuration (6 d) of cadmium. Both effects and kinetics were investigated in systems with and without sediment. Individual Capitella sp. I were exposed to (1) dissolved (i.e. –1). Worms in water-only treatments showed negative growth rates, which decreased linearly …

Evaluation Of Toxicity, Bioavailability And Speciation Of Lead, Zinc And Cadmium In Mine/Mill Wastewaters, Mujde Erten-Unal, Bobby G. Wixson, Nord Gale, Jerry L. Pitt

Civil & Environmental Engineering Faculty Publications

The toxicity of common compounds of lead, cadmium and zinc was evaluated in waters similar to that found in the world's largest lead producing area in Missouri. Static, acute toxicity tests were performed using fathead minnows (Pimephales promelas) and water fleas (Daphnia magna) respectively. Test organisms were subjected to varying amounts of sulfide, carbonate, chloride and sulfate salts of lead, zinc and cadmium mixed in hard, alkaline waters typical to this region. Median lethal concentrations were calculated using nominal versus measured metal concentrations. Measured metal concentrations included four different metal fractionation (extraction/filtration) techniques at different pH …

Ec95-273 Swine Nutrition Guide, Duane Reese, Robert C. Thaler, Mike Brumm, Austin J. Lewis, Philip S. Miller, George W. Libal

University of Nebraska-Lincoln Extension: Historical Materials

This publication is a revision of the previous swine nutrition publication prepared by the Universityof Nebraska and South DakotaState University. The focus of the publication continues to be on nutrient recommendations for swine. Specific factors (nutritional, environmental and managerial) that affect nutrient recommendations have been considered and discussed. We believe that the identification and description of the factors provide the framework for the nutrient recommendations presented inTables 11 to 16. Industry advisors representing various facets of the pork industry were recruited to review and challenge the concepts incorporated in this publication. Also, in situations where “gray areas” existed, these industry …

Bioavailability Of Calcium In Sun-Cured Alfalfa Meal And Effect Of Dietary Calcium Concentration On Bone And Plasma Characteristics During Two Phases Of Gestation In Gilt, G. L. Walker, D. M. Danielson, E. R. Peo, Jr., R. F. Mumm

Department of Animal Science: Faculty Publications

A study was conducted with gravid gilts to determine the bioavailability of Ca in sun-cured alfalfa meal (AM) and the effect of dietary Ca concentration on bone and blood characteristics during two phases of gestation.