Degree Type

Dissertation

Date of Award

2016

Degree Name

Doctor of Philosophy

Department

Food Science and Human Nutrition

Major

Nutritional Sciences

First Advisor

Wendy S. White

Abstract

Vitamin A (VA) is an essential fat-soluble nutrient needed by all vertebrates and plays a central role in essential physiological functions including normal growth, development, immune function, epithelial integrity, reproduction, and vision. Vitamin A deficiency (VAD) is a significant public health problem in more than half of countries worldwide. VAD is a major cause of preventable childhood blindness and also impairs immune function, especially affecting the poorest populations in low and middle-income countries. Based on the World Health Organization (WHO) threshold definition for biochemical VAD (serum retinol below 0.7 µmol/L), most countries in Sub-Saharan Africa and East Asia have over 20% of preschool-age children suffering from VAD. Biofortification is the technique of using plant breeding and/or biotechnology to produce micronutrient-enhanced staple food crops. Because most populations in these areas heavily rely on plant-based diets, provitamin A biofortification is a promising approach to alleviate widespread VAD.

Sorghum is the fourth most important cereal crop worldwide and in Africa. Approximately 300 million of Africa’s most food insecure people who live in the semi-arid tropics rely on sorghum as their staple crop. To alleviate the prevalence of VAD in these areas, the Africa Biofortified Sorghum (ABS) initiative was founded to develop β-carotene-biofortified sorghum through genetic engineering. Our objective was to quantify the bioefficacy of the biofortified sorghum using a Mongolian gerbil model.

Gerbils were fed a VA-free control diet (45% by wt non-transgenic sorghum flour) for 4 wk; 8 animals were then killed at baseline. The remaining animals were randomly assigned to treatments for 6 wk (n = 11 per group): 1) control diet dosed daily with cottonseed oil vehicle; 2) biofortified diet (45% by wt transgenic sorghum flour) (99.6 nmol retinol equivalents/d) dosed daily with cottonseed oil vehicle; 3) control diet dosed daily with β-carotene (84.9 nmol retinol equivalents/d); 4) control diet dosed daily with VA (85.1 nmol retinol equivalents/d). Liver VA stores in the baseline (1.27 ± 0.29 µmol), β-carotene-dosed (1.30 ± 0.26 µmol), and biofortified (1.31 ± 0.23 µmol) groups were not different; liver VA stores in the vehicle-dosed control group were lower (0.92 ± 0.22 µmol) (P < 0.01). Liver VA stores were highest in the VA-dosed group (2.48 ± 0.23 µmol) (P < 0.0001). The calculated bioconversion efficiency for the β-carotene in the biofortified sorghum (4.5 µg β-carotene to 1 µg retinol) was similar to that of the β-carotene dose (3.8 µg to 1 µg retinol). The β-carotene and the other provitamin A carotenoids in the biofortified sorghum: 1) effectively restored liver VA stores in a VA-depleted animal model; 2) had efficacy similar to that of a supplemental β-carotene dose in maintaining liver VA stores.

Accurate quantification of hepatic retinol and retinyl esters is necessary for the determination of vitamin A status in animal models. Our objective for the second study was to optimize methods for the extraction and HPLC analysis of hepatic retinol and retinyl esters.

Our results indicated that Method #1 (J Lipid Res 2014;55:1077-86) resulted in higher hepatic total vitamin A concentrations (423 ± 72.9 nmol/g) than Method #2 (Methods Mol Biol 2010;652:263-75) (36.8 ± 5.35 nmol/g), Method #3 (Anal Methods 2010;2:1320-1332) (347 ± 26.6 nmol/g), and Method #4 (Food Chem 2014;159:477-85) (288 ± 49.1 nmol/g) (P < 0.0001). Of the many factors evaluated within the hepatic VA extraction protocol, an adequate concentration of ethanol (at least 50%) in the homogenization media is necessary for exhaustive extraction. Ethanol is needed in the homogenization media to extract retinoids from liver tissues and then partition them directly to hexane. Homogenizing liver in 100% PBS might facilitate liver tissue binding with water molecules and therefore interfere with the above partitioning process. The initial extraction of VA from the tissue into ethanol is a critical step that needs to be facilitated by an adequate duration of vortexing. Finally, the selection of the reconstitution solvent is important for optimal chromatography and therefore for accurate quantification.

Copyright Owner

Hong You

Language

en

File Format

application/pdf

File Size

129 pages

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