A. Situation or Problem The U.S. population has a deficit of dietary Ca, with 55% of men and 78% of women in the United States not meeting the US recommended daily allowance (RDA) for Ca of 1000 mg/d, putting them at risk for developing osteoporosis. One in two women and one in eight men over the age of 50 will have an osteoporosis-related fracture, resulting in more than $10 billion in health care costs annually. Because a low level of Ca in the diet is among the most important controllable causes of osteoporosis, early intervention in diet is crucial to addressing this important health problem. In the US, approximately 70% of dietary Ca is provided by dairy products such as milk and cheese, with only spinach, collards, or turnip greens as "good" sources of Ca. None of these vegetables forms a significant part of the average diet; however, a more popular vegetable such as lettuce provides only < 2% of the RDA for Ca per cup. Changing individual dietary preferences, for example to include different foods rich in Ca, is a slow and expensive process. In contrast, increasing the levels of vital minerals in dietary staples - such as iodine in salt, fluorine in drinking water, vitamin D in milk, and Ca and niacin in wheat flour - has been a rapid, cost-effective means of changing nutrition on a large scale. From this standpoint, vegetables represent an attractive means of providing increased nutrition to the public since their consumption for health purposes is already encouraged through information campaigns by public health organizations and several low-Ca vegetables (e.g., lettuce) are consumed at much higher levels than Ca-rich vegetables, increasing their value for delivery of increased nutrition. Aside from its large-scale production and consumption, lettuce is an attractive dietary option for enhancing consumption of dietary Ca because it is rich in vitamin K, a co-factor for Ca use in the body, and that one out of four women eat lettuce on any given day, showing that this vegetable is readily consumed by a major at-risk group for osteoporosis. B. Purpose This SBIR Phase I project proposes to develop a novel method of increasing Ca levels in lettuce, a vegetable that comprises a significant part of the average U.S. diet, by using a combination of genetic engineering and low-cost amendments. This Phase I project will demonstrate that these techniques can increase Ca uptake in lettuce to the point that it qualifies as a "good" dietary source of this mineral under USDA guidelines. Successful completion of the project should lay the foundation for introduction of a new value-added crop that provides better nutrition for consumers and a new source of income for agricultural producers. Given the scope of the U.S. dietary Ca deficit, it is highly likely that enhanced Ca content in popular vegetables such as lettuce could measurably improve the health of significant numbers of Americans. In this regard, Ca-fortified lettuce can be viewed as a dietary option for consumers who seek to elevate their intake of Ca without the need to replace favorite foods or rely on mineral supplements. OBJECTIVES: This SBIR Phase I project proposes to develop a novel method of increasing calcium (Ca) levels in two popular lettuce varieties that already comprise a significant part of the average U.S. diet, by combining the use of genetic engineering with low-cost hydroponic amendments. Currently, additional dietary calcium has been provided in the form of nutritional supplements; however, most of these supplements, which many consumers fail to take regularly, provide the calcium in a non-bioavailable, inorganic form that fails to be adequately metabolized in the human body. Increasing the calcium content of a popular vegetable that provides the additional calcium in bioavailable form will help to protect consumers against the onset of osteoporosis. The proposed research will address the following objectives: (i) Demonstrate that hydroponic chemical amendments, applied to transgenic lettuce, increase calcium content relative to control treatment by a factor of 2 or more, to the point that a single helping of lettuce (i.e., one cup) would contain about 250 mg of Ca, equivalent to 25% of the RDA for calcium per serving and sufficient to qualify as a "good" source of calcium by USDA, and (ii) Demonstrate good (75%) in vitro bioavailability of Ca in the transformed lettuce produced in (i). If successful, the enhanced lettuce plants will help to increase consumption of dietary calcium by consumers believed to be at risk for osteoporosis (e.g., post-menopausal women) as well as increase overall calcium levels in the diets of other consumers to take advantage of the bone-strengthening properties associated with higher levels of calcium intake. This research should provide the basis for a cost-effective, grower-based technology that increases the nutritional value of a food staple and which has a large commercial market in the U.S. and abroad
