Photosynthesis of vitamin D in the skin: effect of environmental and life-style variables

Exposure to sunlight continues to play a major role in providing adequate vitamin D nutrition for most of the population of the world, including those who live in countries that practice fortification of dairy, margarine, and cereal products with vitamin D. During exposure to sunlight, the high-energy UV photons (290-315 nm) penetrate the epidermis and photolyze 7-dehydrocholesterol (provitamin D3) to previtamin D3. Once formed, previtamin D3 undergoes a thermally induced isomerization to vitamin D3 that takes 2-3 days to reach completion. Melanin effectively competes with provitamin D3 for the UV radiation that enters the epidermis and limits its photolysis to previtamin D3. However, this is not the major factor that prevents excess production of vitamin D in the skin of people who are constantly exposed to sunlight. During the initial exposure to sunlight, provitamin D3 is efficiently converted to previtamin D3. However, because previtamin D3 is photolabile, continued exposure to sunlight causes the isomerization of previtamin D3, principally to lumisterol. Thus, no more than 10-20% of the initial provitamin D3 concentrations ultimately end up as previtamin D3. Aging, sunscreens, seasonal changes, time of day, and latitude also significantly affect the cutaneous production of this vitamin-hormone.     

Vitamin D supplementation increases the attractiveness of males' scent for female Iberian rock lizards

Evolutionary theory proposes that signals used in sexual selection can only be stable if they are honest and condition dependent. However, despite the fact that chemical signals are used by many animals, empirical research has mainly focused on visual and acoustic signals. Vitamin D is an essential nutrient for lizards, but in some lizards its precursor (cholesta-5,7-dien-3-ol=provitamin D) is found in femoral gland secretions, which males use for scent marking and intraspecific communication. By allocating provitamin D to secretions, males might need to divert vitamin D from metabolism. This might be costly and condition dependent. We tested whether diet quality affected chemical signals of male Iberian rock lizards (Lacerta monticola) and its consequences for sexual selection. After experimental supplementation of dietary vitamin D, males increased the proportion of provitamin D in femoral secretions. Further experiments showed that females detected these changes in males' signals by chemosensory cues, and discriminated provitamin D, and changes in its concentration, from similar steroids (i.e. cholesterol) found in secretions. Moreover, females preferred areas scent marked by males with more provitamin D in their secretions. This mechanism would confer honesty to chemical signals of male lizards, and, thus, females may rely on it to select high-quality males. We suggest that the allocation of vitamins and other essential nutrients to either visual (e.g. carotenoids) or chemical ornaments might be the common basis of honest sexual displays in many animals.     

Two methods for direct assessment of the Vitamin D synthetic capacity of sunlight and artificial UV sources

Excessive UV exposures are commonly associated with adverse health effects, but proper amounts of UV are beneficial for people and essential in the natural production of Vitamin D(3) in skin. Two methods have been developed for direct evaluation of the Vitamin D synthetic capacity of sunlight (and artificial UV sources). The first one uses an in vitro model of Vitamin D(3) synthesis (ethanol solution of 7-dehydrocholesterol, 7-DHC), and concentration of previtamin D(3) accumulated during an UV exposure is determined using specially designed spectrophotometric analysis. The second method utilizes photoisomerization of provitamin D in nematic liquid crystalline (LC) matrix, and visual estimation of accumulated previtamin D becomes possible due to special design of a LC cell. This user-friendly method is appropriate for personal UV dosimetry and may have wide application in tanning saloons, in clinical dermatology and UV therapy.     

The induced biosynthesis of 7-dehydrocholesterols in yeast: potential sources of new provitamin D3 analogs.

The effect of low concentrations of a specifically designed sterol-24-transmethylase inhibitor, 25-aza-24, 25-dihydrozymosterol (10) on sterol production in Saccharomyces cerevisiae was examined. The synthesis of cholesta-5,7,22,24-tetraen-3beta-ol (4), its 7,22,24 analog (15) and the 7,24 analog (5) coupled with the availability of zymosterol (6) and cholesta-5,7,24-3beta-ol (3) derivatives facilitated a search for these sterols in cultures treated with this inhibitor. When S. cerevisiae was grown in the presence of 1.3 and 5 muM 10, it produced no ergosterol but accumulated zymosterol (6), cholesta-5,7,22,24-tetraen-3beta-ol (4) and related C27 sterols (3 and 5). These results indicate blockage of the side chain methylation that normally occurs during the biosynthesis of ergosterol in yeast by compound 10 is efficient. The cholesta-5,7,22,24-tetraen-3beta-ol is a close structural analog of provitamin D3 (7-dehydrocholesterol). The inhibited yeast thus provides a source of a potentially new provitamin D3 substitute.     

Is provitamin D a UV-B receptor in plants?

An hypothesis is presented that provitamin D (dehydrocholesterol and/or ergosterol) can act as a UV-B receptor in plants and algae. We also propose that the proportions between provitamins D, previtamins D, and vitamins D (D₂ and D₃), after calibration, can be used to evaluate UV-B exposure of phytoplankton and terrestrial vegetation.     

A computational model for previtamin D(3) production in skin

Low levels of vitamin D have been implicated in a wide variety of health issues from calcemic diseases to cancer, diabetes and cardiovascular disease. For most humans, the majority of vitamin D(3) is derived from sunlight. How much vitamin D is produced under given exposure conditions is still widely discussed. We present a computational model for the production of (pre-)vitamin D within the skin. It accounts for spectral irradiance, optical properties of the skin and concentration profile of provitamin D. Results are computed for various sets of these parameters yielding the distribution of produced previtamin D in the skin.     

Historic reflection on steroids: Lederle and personal aspects.

Steroid research at Lederle was initiated in 1946 and continued until about 1971. The areas of research interest involved primarily a study of delta 5,7-steroids, provitamin D3 variants, dehydroepiandrosterone variants, steroidal ketals, corticoids, and steroid conjugates. One of the principal goals was to develop novel antiinflammatory steroids for the treatment of, e.g., rheumatoid arthritis. A rational design led to the development of 16 alpha-hydroxycorticoids, culminating in the synthesis and therapeutic use of triamcinolone and related compounds.     

Metabolic engineering of potato tuber carotenoids through tuber-specific silencing of lycopene epsilon cyclase

Background  

Potato is a major staple food, and modification of its provitamin content is a possible means for alleviating nutritional deficiencies. beta-carotene is the main dietary precursor of vitamin A. Potato tubers contain low levels of carotenoids, composed mainly of the xanthophylls lutein, antheraxanthin, violaxanthin, and of xanthophyll esters. None of these carotenoids have provitamin A activity.     

Provitamin A accumulation in cassava (Manihot esculenta) roots driven by a single nucleotide polymorphism in a phytoene synthase gene

Cassava (Manihot esculenta) is an important staple crop, especially in the arid tropics. Because roots of commercial cassava cultivars contain a limited amount of provitamin A carotenoids, both conventional breeding and genetic modification are being applied to increase their production and accumulation to fight vitamin A deficiency disorders. We show here that an allelic polymorphism in one of the two expressed phytoene synthase (PSY) genes is capable of enhancing the flux of carbon through carotenogenesis, thus leading to the accumulation of colored provitamin A carotenoids in storage roots. A single nucleotide polymorphism present only in yellow-rooted cultivars cosegregates with colored roots in a breeding pedigree. The resulting amino acid exchange in a highly conserved region of PSY provides increased catalytic activity in vitro and is able to increase carotenoid production in recombinant yeast and Escherichia coli cells. Consequently, cassava plants overexpressing a PSY transgene produce yellow-fleshed, high-carotenoid roots. This newly characterized PSY allele provides means to improve cassava provitamin A content in cassava roots through both breeding and genetic modification.     

The photobiogenesis and metabolism of vitamin D.

Provitamin D3 (7-dehydrocholesterol) is converted to previtamin D3 by the action of ultraviolet radiation on the skin. Previtamin D3 thermally isomerizes to vitamin D3 in the skin and the vitamin is then transported to the liver on the vitamin D-binding protein. Although there are extrahepatic vitamin D-25-hydroxylases, the liver is the major site for the 25-hydroxylation of vitamin D. In response to hypocalcemia and hypophosphatemia, 25-OH-D is metabolized by a renal-cytochrome. P450-dependent mixed function oxidase system is 1alpha,25(OH)2D. When calcium and phosphate homeostasis prevails the renal 25-OH-D-1alpha-hydroxylase activity is limited and instead a non-cytochrome P450 mixed function oxidase metabolizes 25-OH-D to 24R,25(OH)2D. Parathyroid hormone has clearly been shown to be a trophin for the renal synthesis of 1,25(OH)2D; however, the role and significance of the adrenal steroids, or gonadal and pituitary hormones, on the renal 25-OH-D-1alpha-hydroxylase is not well defined. The regulation of the photometabolism of provitamin D3 to vitamin D3, the role and significance of the side-chain metabolism of 1,25(OH)2D by the small intestine, and the metabolism of 25-OH-D to 24R,25(OH)2D by chondrocytes and its stimulation of protein synthesis in these cells are just a few issues that will require further investigation.     

Provitamin A conversion to retinal via the beta,beta-carotene-15,15'-oxygenase (bcox) is essential for pattern formation and differentiation during zebrafish embryogenesis

The egg yolk of vertebrates contains carotenoids, which account for its characteristic yellow color in some species. Such plant-derived compounds, e.g. beta-carotene, serve as the natural precursors (provitamins) of vitamin A, which is indispensable for chordate development. As egg yolk also contains stored vitamin A, carotenoids have so far been solely discussed as pigments for the coloration of the offspring. Based on our recent molecular identification of the enzyme catalyzing provitamin A conversion to vitamin A, we address a possible role of provitamin A during zebrafish (Danio rerio) development. We cloned the zebrafish gene encoding the vitamin A-forming enzyme, a beta,beta-carotene-15,15'-oxygenase. Analysis of its mRNA expression revealed that it is under complex spatial and temporal control during development. Targeted gene knockdown using the morpholino antisense oligonucleotide technique indicated a vital role of the provitamin A-converting enzyme. Morpholino-injected embryos developed a morphological phenotype that included severe malformation of the eyes, the craniofacial skeleton and pectoral fins, as well as reduced pigmentation. Analyses of gene expression changes in the morphants revealed that distinct retinoic acid-dependent developmental processes are impaired, such as patterning of the hindbrain and differentiation of hindbrain neurons, differentiation of neural crest derivatives (including the craniofacial skeleton), and the establishment of the ventral retina. Our data provide strong evidence that, for several developmental processes, retinoic acid generation depends on local de novo formation of retinal from provitamin A via the carotene oxygenase, revealing an unexpected, essential role for carotenoids in embryonic development.     

Generation of transgenic maize with enhanced provitamin A content

Vitamin A deficiency (VAD) affects over 250 million people worldwide and is one of the most prevalent nutritional deficiencies in developing countries, resulting in significant socio-economic losses. Provitamin A carotenoids such as beta-carotene, are derived from plant foods and are a major source of vitamin A for the majority of the world's population. Several years of intense research has resulted in the production of 'Golden Rice 2' which contains sufficiently high levels of provitamin A carotenoids to combat VAD. In this report, the focus is on the generation of transgenic maize with enhanced provitamin A content in their kernels. Overexpression of the bacterial genes crtB (for phytoene synthase) and crtI (for the four desaturation steps of the carotenoid pathway catalysed by phytoene desaturase and zeta-carotene desaturase in plants), under the control of a 'super gamma-zein promoter' for endosperm-specific expression, resulted in an increase of total carotenoids of up to 34-fold with a preferential accumulation of beta-carotene in the maize endosperm. The levels attained approach those estimated to have a significant impact on the nutritional status of target populations in developing countries. The high beta-carotene trait was found to be reproducible over at least four generations. Gene expression analyses suggest that increased accumulation of beta-carotene is due to an up-regulation of the endogenous lycopene beta-cylase. These experiments set the stage for the design of transgenic approaches to generate provitamin A-rich maize that will help alleviate VAD.     

An LC/MS method for d8-β-carotene and d4-retinyl esters: β-carotene absorption and its conversion to vitamin A in humans

The intestinal absorption and metabolism of β-carotene is of vital importance in humans, especially in populations that obtain the majority of their vitamin A from provitamin A carotenoids. MS has provided a better understanding of the absorption of β-carotene, the most potent provitamin A carotenoid, through the use of stable isotopes of β-carotene. We report here an HPLC-MS method that eliminates the need for complicated sample preparation and allows us to detect and quantify newly absorbed d8-β-carotene as well as its d4-retinyl ester metabolites in human plasma and chylomicron fractions. Both retinoids and β-carotene were recovered in a single simple extraction that did not involve saponification, thus allowing subsequent quantitation of individual fatty acyl esters of retinol. Separation of d8-β-carotene and its d4-retinyl ester metabolites was achieved using the same C30 reversed-phase liquid chromatography followed by mass spectrometry in selected ion monitoring and negative atmospheric pressure chemical ionization modes, respectively. Total time for the two successive runs was 30 min. This HPLC-MS method allowed us to quantify the absorption of intact d8-β-carotene as well as its extent of conversion to d4-retinyl esters in humans after consumption of a single 5 mg dose of d8-β-carotene.     

Ultraviolet exposure and vitamin D synthesis in a sun-dwelling and a shade-dwelling species of Anolis: are there adaptations for lower ultraviolet B and dietary vitamin D3 availability in the shade?

We compared the natural ultraviolet B (UV-B) exposure, dietary vitamin D, and skin-generated vitamin D synthesis for adult males of two species of Jamaican anoles. The more shade-tolerant and thermal-conforming Anolis lineotopus merope, rarely exposed to full sun, experienced less UV-B irradiation in its shady environment than the more heliophilic and thermophilic Anolis sagrei, which frequently basked in full sun during the morning hours (0800-1100 hours). Both species obtained detectable levels of vitamin D(3) in their diet, but the heliophilic A. sagrei obtained more. To compensate for less availability of UV-B and dietary vitamin D, the skin of A. lineotopus merope seems to have acquired a greater sensitivity than that of A. sagrei regarding UV-B-induced vitamin D(3) photobiosynthesis. We assessed this by observing a greater conversion of provitamin D to photoproducts in skin exposed to UV-B from a sunlamp. The reduced skin sensitivity of A. sagrei regarding vitamin D photobiosynthesis may reflect a correlated response associated with less need for vitamin D photobiosynthesis and greater need for UV-B screening capacity as an adaptation to a more damaging UV-B environment. However, the possibility that adaptations for photobiosynthesis of vitamin D and for protection from skin damage could involve independent mechanisms needs investigation. Also, the ability to behaviorally regulate UV-B exposure, as shown for the panther chameleon, would benefit both species of Anolis and should be investigated.     

The effect of ergosterol, ergocalciferol and cholecalciferol on calcium-controlled peroxidase secretion by sugarbeet cells

Provitamin D₂ (ergosterol), vitamins D₂ and D₃ reduced the calcium-mediated peroxidase secretion in three types of sugarbeet cells in suspension cultures. Vitamin D₂ was the most effective in habituated non-organogenic cells which were the less sensitive to calcium; provitamin D₂ was the most effective in habituated organogenic cells, while normal non-organogenic cells were equally sensitive to the three types of vitamin D. The calcium ionophore A23187 slightly restricted peroxidase release in all cases, except in the habituated organogenic cells in the presence of calcium where it exerted a promotive effect. The inhibiting effect of vitamin D₂ was not counteracted by the ionophore except in this habituated organogenic cell line.     

Identification of natural ligands of retinoic acid receptor-related orphan receptor alpha ligand-binding domain expressed in Sf9 cells--a mass spectrometry approach

The ligand-binding domain (LBD) of the human retinoic acid receptor-related orphan receptor (RORalpha-LBD), expressed in Sf9 cells, was purified and analyzed by electrospray ionization-mass spectrometry (ESI-MS). ESI-MS operated under native conditions showed the presence of a fortuitous ligand with molecular weight 386. Further analysis by gas chromatography-mass spectrometry (GC-MS) allowed the identification of the ligands bound to the LBD. Cholesterol (77%) and 7-dehydrocholesterol (provitamin D(3); 18%) were shown to be the major ligands. A monohydroxylated cholesterol derivative was identified as a minor ligand. In addition, ligand exchange experiments monitored by ESI-MS showed that cholesterol sulfate has a higher affinity for RORalpha-LBD than cholesterol and 25-hydroxycholesterol. Binding of coactivator (CoA) peptide GRIP1P was shown to occur in a stoichiometric manner. Therefore, monitoring of binding of CoAs by mass spectrometry could be used for classification of the ligands as agonist or antagonist molecules.     

Separation and quantitation of serum beta-carotene and other carotenoids by high performance liquid chromatography

We describe a specific assay for serum provitamin A (alpha- and beta-carotene) by high pressure liquid chromatography (HPLC). The system separates alpha- and beta-carotene in 7.4 min using a C18 muBondapak, 10-micron particle size column with a mobile phase of acetonitrile-chloroform 92:8 at 2 ml per min and a 462 nm detector. The HPLC assay had a recovery of 94.8% of added beta-carotene and, at a serum concentration of 215.2 micrograms/L, had within-run and between-run precisions of 3.1% and 3.6%, respectively. In 65 subjects, the HPLC-determined provitamin A (alpha- and beta-carotene) value was 343 +/- 166 micrograms/L and averaged 23.4 +/- 7.9% (range 9-43%) of the values obtained by a traditional colorimetric assay for total serum "carotenes." Although total serum carotenes showed no relationship to serum vitamin A (r = -0.048; P = 0.78), HPLC-determined alpha- and beta-carotene was significantly inversely correlated (r = -0.357; P = 0.05).     

Effects of Artificial Ultraviolet Light Exposure on Reproductive Success of the Female Panther Chameleon (Furcifer pardalis) in Captivity

Having previously documented experimentally the need for ultraviolet B (UVB) irradiation (290–315 nm) in the light environment of captive female panther chameleons (Furcifer pardalis) to ensure hatching success of their eggs, we investigated optimal UVB irradiation levels. From 1996–1998 28 hatchling female panther chameleons were raised to maturity and bred (using vitamin and mineral‐fortified insect diets low in vitamin D) in nine different artificial UVB light environments. Seven of the environments included long (12 hr/day) low irradia‐tion exposures ranging from 1.7 to 22 μW/cm² UVB, with a corresponding conversion of provitamin D₃ to photoproducts in in vitro models of 0.18 to 1.52% in 2 hr. Two environments included short (0.5 and 1.0 hr/day), high irradiation exposures of 55 and 49 μW/cm² UVB, respectively, with a corres‐ponding conversion of provitamin D₃ to photoproducts in in vitro models of 8.3% to 14.6% in the respective 0.5‐ and 1.0‐hr time periods. Females raised with the mid‐level long/low exposures (5–15 μW/cm² UVB; 0.52–1.32% conversion to photoproducts in in vitro models) produced viable eggs with a significantly higher percentage of hatching compared to those with the extreme (highest or lowest) long/low exposures. Those raised with the short‐/high‐exposure environments produced viable eggs with a generally high percentage of hatching, but success was variable. The results and techniques for light quality assessment are interpreted, with recommendations for practical application by the herpetoculturist desiring to successfully breed panther chameleons in captivity. Zoo Biol 21:525–537, 2002. © 2002 Wiley‐Liss, Inc.     

Vitamin production in transgenic plants

Plants are a major source of vitamins in the human diet. Due to their significance for human health and development, research has been initiated to understand the biosynthesis of vitamins in plants. The pathways that are furthest advanced in elucidation are those of provitamin A, vitamin C and vitamin E. There is little knowledge about the regulation, storage, sink and degradation of any vitamin made in plants, or the interaction of vitamin biosynthetic pathways with other metabolic pathways. Researchers as well as life science companies have endeavoured to manipulate levels of vitamins in order to create functional food with enhanced health benefits, and even with the goal of achieving levels worth extracting from plant tissues. Thus far, metabolic engineering has resulted in transgenic plants that contain elevated levels of provitamin A, vitamin C and E, respectively. Additional research is necessary to identify all relevant target genes in order to further improve and tailor plants with elevated vitamin contents at will.