Vitamin D in plants : Occurrence, analysis and biosynthesis

Abstract

The major function of vitamin D in vertebrates is maintenance and regulation of calcium homeostasis and vitamin D is, therefore, critically important for development of a healthy skeleton. Thus, vitamin D insufficiency increases the risk of osteoporosis, but has also been linked to increased risk of hypertension, autoimmune diseases, diabetes and cancer. There is consequently a growing awareness about vitamin D as a requirement for optimal health. Vitamin D3 is synthesized in the skin by photochemical conversion of provitamin D3 (7-dehydrocholesterol) by exposure to sunlight at 290-315 nm. However, the necessary wavelengths are not emitted from October to March in Denmark and no vitamin D3 is consequently synthesized in the skin during winter. Unfortunately, very few food sources naturally contain vitamin D and the general population as a result fail to meet their vitamin D requirements. As a surprise for many is vitamin D present in several plants. The hypothesis, which is the background for this PhD thesis, is that plants can be a source of vitamin D for humans as well as for animals. The overall aim was to study the occurrence and biosynthesis of vitamin D in plants to test this hypothesis. Most work on vitamin D in plants has been done with non-selective methods such as bioassays and special emphasis was, therefore, placed on development of analytical methods to study vitamin D, its sterol precursors and hydroxylated metabolites in more details. All developed methods were based on liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) detection, because of the superior selectivity and sensitivity. The developed methods were used in three studies covering various aspects of vitamin D in plants. The term vitamin D includes both vitamin D2 and vitamin D3. The provitamin of vitamin D2 is ergosterol that also is the predominant sterol in fungi. Small amounts of ergosterol can be found in plants contaminated with fungi and the conversion to vitamin D2 occurs by UVBexposure of the plant material during growth. Six varieties of perennial ryegrass (Lolium perenne L.) were harvested four times during the season and analyzed for ergosterol and vitamin D2. The average content of vitamin D2 was 2 µg/kg, which was maximum 2‰ of the ergosterol content. The content of both vitamin D2 and ergosterol changed more than a factor of ten during the season and a combination of sun and precipitation was important for the synthesis of vitamin D2. The synthesis of vitamin D3 in plants is unresolved and contradicting results concerning the dependence of light have been presented. Various plants were consequently exposed to UVB-light during growth and analyzed for vitamin D3. Vitamin D3 was identified in the leaves of Solanum glaucophyllum Desf., Solanum lycopersicum L. and Capsicum annuum L., belonging to the taxonomic family Solanaceae. Vitamin D3 was found in both UVB- and non-UVB-treated plants, but the content of the UVB-treated plants was 18-64 times higher. No vitamin D3 was found in Pisum sativum L. and Sorghum bicolor (L.) Moench belonging to Fabaceae and Poaceae, respectively. It still needs to be fully established how vitamin D3 is formed in plants, but both cholesterol and 7-dehydrocholesterol were found in all vitamin D3 synthesizing plants and may serve as precursors of vitamin D3 in plants. Vitamin D is biologically inactive and activation involves two hydroxylations. Vitamin D is first hydroxylated in the liver to 25-hydroxyvitamin D and subsequently to 1,25-dihydroxy vitamin D3 in the kidneys. An enzymatic pathway similar to that in animals may be present in plants, since enzymatic activity involved in formation of 25OHD3 and 1,25(OH)2D3 earlier has been identified in Solanum glaucophyllum. The hydroxylated metabolite, 25-hydroxy vitamin D3, was identified in Solanum lycopersicum, Capsicum annuum and Solanum glaucophyllum. The dihydroxylated metabolite, 1,25-dihydroxy vitamin D3, was only found in Solanum glaucophyllum. Enzymatic hydrolysis was used to study the occurrence of glycoside conjugates. These were found exclusively for 1,25-dihydroxy vitamin D3 in UVB-treated Solanum glaucophyllum. Altogether, this PhD thesis has shown that both vitamin D2 and vitamin D3 can be found in plants. The results demonstrate that grass potentially can be a significant source of vitamin D for grazing animals and animals fed on silage and hay. Especially, leaves from the Solanaceous family, where potato and tomato belong, contain high amounts of not only vitamin D3, but also the hydroxylated metabolites of vitamin D3. The presence of the hydroxylated metabolites is of particular interest because the activity is believed to be 5-10 times that of vitamin D3. Further studies are needed to determine if also the fruits contain vitamin D3. These studies may help to determine whether plants have a potential as a new source of vitamin D.