Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
Nutritional Sciences
Non Technical Summary
Excessive dietary levels of vitamin A, leading to intoxication, have negative consequences on bone health. However, the effects of hypervitaminosis A without overt clinical signs are less defined, especially in conjunction with suboptimal vitamin D status, now considered a pandemic in the world. Conversely, vitamin A deficiency, which leads to impaired growth is widespread in low- and middle-income countries in Southeast Asia and sub-Saharan Africa. The lack of sensitive vitamin A status monitoring programs, coupled with overlapping interventions to treat vitamin A deficiency (e.g., staple food fortification, high-dose supplementation), have resulted in a high prevalence of hypervitaminosis A in some cohorts, negatively impacting bone turnover serum biomarkers. In a recent study in the United States, vitamin A deficiency and hypervitaminosis A, including toxicity evaluated by liver evaluations, were reported in adults at alarmingly high prevalence. Conclusive data regarding specific bone health outcomes related to hypervitaminosis A due to elevated dietary intake, and its interactions with different vitamin D and calcium statuses, are needed to shape current and future nutritional guidelines regarding vitamin A interventions and food formulation. The studies proposed will investigate the influence of excessive levels of vitamin A and modulation of vitamin D and calcium intakes in a pig model. Bone abnormalities will be analyzed by clinically translatable imaging techniques in a swine model of human vitamin A and D and calcium metabolism. These studies will also yield useful data on formulations for feed in the swine industry.
Animal Health Component
25%
Research Effort Categories
Basic
50%
Applied
25%
Developmental
25%
Goals / Objectives
Hypotheses: We hypothesize that excessive vitamin A, especially in combination with vitamin D deficiency will lead to bone abnormalities, and these effects will be rescued by providing excess dietary calcium.Supporting Objectives-The proposed studies have two overarching objectives that will use innovative methods to assess:1) The interactions of non-optimal (deficient or excessive) vitamin A and D statuses on growth and bone development in young growing pigs.We will measure bone turnover biomarkers, growth, and quality by dual-energy X-ray absorptiometry (DXA) and clinical-computed tomography (CT) of pigs fed combinations of deficient or excessive levels of vitamins A and D.We will determine how vitamin D deficiency and hypervitaminosis interact with excessive levels of vitamin A to worsen or mitigate its impact on bone metabolism.2) The effect of suboptimal (deficient or excessive) dietary vitamin A on a background of adequate and excessive calcium intake.We will modulate dietary vitamin A and calcium to determine changes in detrimental or protective clinical outcomes in growing pigs. Adequate vitamin D will be provided.These studies could prove to be relevant in animals due to the tendency of commercial swine feed manufacturers to add nutrients at levels far above NRC recommendations, and in humans where overlapping interventions for vitamin A deficiency and excessive intake of preformed retinol due to supplements and fortification can lead to hypervitaminosis A. Balancing vitamins A and D and calcium are important food safety consideration.
Project Methods
Detailed Methods and Techniques to be Employed in this Project:Techniques will be identical in both studies, unless further relevant analyses are identified during the course of Study 1 to be applied to Study 2.Gross observations, body weight, and feed consumption will be measured. Basic pathologies (e.g., rickets, lameness, failure to grow, diarrhea) will be noted throughout the studies. Serum, liver, kidney, and adipose will be analyzed for retinol and retinyl esters using on-going HPLC and ultra-HPLC (UPLC) analyses with methods in PD Tanumihardjo's laboratory.Bone mineral density, total mineral content, and bone area will be analyzed using a clinical (human-sized) DXA instrument. The pigs will be anesthetized and scanned to determine differences in whole-body mineral density, a significant improvement over previous bone ashing techniques due to its comprehensive nature and translation of results to living human subjects or animals in future research. Co-PD Crenshaw has experience performing this analysis on living swine.Extensive bone and growth plate analysis by CT using 3-dimensional analysis techniques will be used as developed by co-PD Crenshaw. At kill, femurs will be excised from the carcass and 2-4 cm muscle tissue will be retained to improve CT-scan image quality. Femurs will be coded and individually stored at 4°C until evaluated by CT. Femurs will be brought to room temperature before being scanned using a CT instrument (GE Lightspeed 16, GE Medical System; Waukesha, WI). An exposure setting of 120 kV will be used with 30 mA in a helical mode. Scans are made at a 0.625 mm slice thickness and a field of view of 20 cm. Image resolution is 512 x 512, with a pixel size of 0.391 mm (area 0.153 mm2) and voxel volume 0.096 mm3 (0.391 x 0.391 x 0.625 mm). A subjective score for observed bone anomalies will be assigned based on examination of the excised femur joint surface. This non-destructive analysis, which was previously used to locate and measure osteochondral lesions by co-PD Crenshaw's lab will yield quantitative information using a method that could be applied to humans in clinics. Images from CT scans of excised femurs will be reconstructed using Mimics software (Materialize 19.0) to measure the number, volume, and surface area of lesions across longitudinal sections of the epiphyseal growth plate region in the distal femur CT scan image. Because early lesions of osteochondrosis are exhibited by regions of cartilage retention along the growth plate, the lack of mineralized tissue can be detected by void regions in x-ray attenuation. Masks of non-bone cavities along the diaphyseal region next to the growth plate will be applied to determine the number, volume, surface area, and location of the lesion-like disruptions in normal bone patterns. This approach will allow 3D and anatomical distribution assessments of individual lesions. Regions of interest can be used to determine bone density and trabecular structure properties, including cortical bone volume, thickness, pore density, and periosteal and endosteal perimeters, any of which could be affected by hypervitaminosis A based on its negative effect on bone mineral density.Following CT, mechanical strength of the bones will be determined by a four-point bending test. These data will provide insight into the effects of hypervitaminosis A on the propensity for bone fracture in humans.Transcript levels of relevant bone-metabolism enzymes (e.g., osteoprotegerin, RANKL, and RANK) and markers of osteoclast, osteoblast, and osteocyte proportions and development will be probed in ground bone by routine RT-qPCR to identify links from dietary group to bone outcomes as previously completed in co-PD Crenshaw's laboratory.Dissected tissues for gene expression will be frozen immediately in liquid nitrogen and stored at -80°C. Frozen tissues will be homogenized in Trizol Reagent (Life Technologies) and RNA isolated as per the manufacturer's instructions. Isolated total RNA (1 mg) will be treated with DNase, reverse transcribed using the High Capacity cDNA Kit (Applied Biosystems), and then diluted to 100 μL with RNase/DNase-free water. Quantitative (q)-PCR will be performed using primers specific to a select set of differentially expressed genes by Taqman analyses. TaqMan Gene Expression probes (Applied Biosystems) will be used for reverse transcriptase-PCR (RT-PCR).Vitamin D status and metabolism among groups will be analyzed as follows:Serum 25-(OH)-D and 24,25-(OH)2D will be analyzed by UPLC (Waters, Milford, MA) as previously described, with modifications to resolve 24,25-(OH)2D. Building on published liquid chromatography coupled to tandem mass spectrometry methods co-PD Pike has previously used in his research, we will seek assistance to analyze a subset of the piglets' serum for other vitamin D metabolites by to determine differences in vitamin D status [as 25-(OH)-D], activation [1,25-(OH)2-D], and degradation [24,25-(OH)2-D and 1,24,25-(OH)3-D].Renal mRNA transcript levels of the vitamin D activation enzyme 1a-hydroxylase CYP27B1 and 24-hydroxylase degradation enzyme CYP24A1 will be analyzed by RT-qPCR under supervision of co-PD Pike's laboratory staff, who are experts in regulation of these genes. Both 1,25-(OH)2-D and retinoic acid are known transcriptional regulators of CYP24A1, providing one potential site of interaction.Calcium and phosphorus will be analyzed by inductively coupled plasma optical emission spectroscopy. The calcium- and phosphorus-regulating serum parathyroid hormone (calcium-regulating hormone, PTH), fibroblast growth factor 23 (phosphorus regulation, FGF23), C-terminal telopeptide of type I collagen (bone resorption, CTX-1), and N-terminal propeptide of type I procollagen (bone formation, P1NP) will be measured by enzyme-linked immunosorbent assay or radioimmunoassay (if appropriate) under co-PD Pike's supervision.As with vitamin D, vitamin A status and metabolism will be analyzed in serum and liver (the site of storage of 80-95% of vitamin A in healthy individuals) by UPLC of metabolites, and RT-qPCR of the liver vitamin A storage enzyme LRAT and degradation enzyme CYP26A1. The Tanumihardjo Vitamin A Assessment lab has completed and published numerous studies with vitamin A assessment from pig tissues.