Tagging of biomolecules with deuterated water (D2O) in commercially important microalgae.

To understand the effect of any biomolecules in specific metabolic pathways in humans, bioavailability and for other basic understanding, stable isotopically-labelled biomolecules (preferably deuterated) is the fundamental pre-requisite. Production of deuterated biomolecules such as, astaxanthin, ß-carotene, lutein, chlorophyll-a, and eicosapentaenoic acid (EPA, 20:5n-3) by metabolic tagging have been shown in commercially important microalgae, Haematococcus pluvialis and Phaeodactylum tricornutum. These microalgae were grown in appropriate optimized medium supplemented with 25% (v/v) deuterated water. LC-MS analysis showed a maximum of 20, 25, 23, 24, and 27% replacement of hydrogen by deuterium atoms respectively in astaxanthin, ß-carotene, lutein, chlorophyll-a, and EPA. To our knowledge, this is the first report on the production of deuterated astaxanthin, chlorophyll-a and EPA by these microalgae.

Techniques for measuring vitamin A activity from ß-carotene.

Dietary ß-carotene is the most important precursor of vitamin A. However, the determination of the efficiency of in vivo conversion of ß-carotene to vitamin A requires sensitive and safe techniques. It presents the following challenges: 1) circulating ß-carotene concentration cannot be altered by eating a meal containing ≤6 mg ß-carotene; 2) because retinol concentrations are homeostatically controlled, the conversion of ß-carotene into vitamin A cannot be estimated accurately in well-nourished humans by assessing changes in serum retinol after supplementation with ß-carotene. In the past half-century, techniques using radioisotopes of ß-carotene and vitamin A, depletion-repletion with vitamin A and ß-carotene supplements, measurement of postprandial chylomicron fractions after consumption of a ß-carotene dose, and finally, stable isotopes as tracers to follow the absorption and conversion of ß-carotene in humans have been developed. The reported values for ß-carotene to vitamin A conversion showed a wide variation from 2 µg ß-carotene to 1 µg retinol (for synthetic pure ß-carotene in oil) and 28 µg ß-carotene to 1 µg retinol (for ß-carotene from vegetables). In recent years, a stable isotope reference method (IRM) was developed that used labeled synthetic ß-carotene. The IRM method provided evidence that the conversion of ß-carotene to vitamin A is likely dose dependent. With the development of intrinsically labeled plant foods harvested from a hydroponic system with heavy water, vitamin A activity of stable isotope-labeled biosynthetic ß-carotene from various foods consumed by humans was studied. The efficacy of plant foods rich in ß-carotene, such as natural (spinach, carrots, spirulina), hybrid (high-ß-carotene yellow maize), and bioengineered (Golden Rice) foods, to provide vitamin A has shown promising results. The results from these studies will be of practical importance in recommendations for the use of pure ß-carotene and foods rich in ß-carotene in providing vitamin A and ultimately in preventing either overconsumption or poor intake of vitamin A by humans.

ß-Carotene in Golden Rice is as good as ß-carotene in oil at providing vitamin A to children.

BACKGROUND: Golden Rice (GR) has been genetically engineered to be rich in ß-carotene for use as a source of vitamin A. OBJECTIVE: The objective was to compare the vitamin A value of ß-carotene in GR and in spinach with that of pure ß-carotene in oil when consumed by children. DESIGN: Children (n = 68; age 6-8 y) were randomly assigned to consume GR or spinach (both grown in a nutrient solution containing 23 atom% ²H2O) or [²H8]ß-carotene in an oil capsule. The GR and spinach ß-carotene were enriched with deuterium (²H) with the highest abundance molecular mass (M) at M(ß-C)+²H10. [¹³C10]Retinyl acetate in an oil capsule was administered as a reference dose. Serum samples collected from subjects were analyzed by using gas chromatography electron-capture negative chemical ionization mass spectrometry for the enrichments of labeled retinol: M(retinol)+4 (from [²H8]ß-carotene in oil), M(retinol)+5 (from GR or spinach [²H10]ß-carotene), and M(retinol)+10 (from [¹³C10]retinyl acetate). RESULTS: Using the response to the dose of [¹³C10]retinyl acetate (0.5 mg) as a reference, our results (with the use of AUC of molar enrichment at days 1, 3, 7, 14, and 21 after the labeled doses) showed that the conversions of pure ß-carotene (0.5 mg), GR ß-carotene (0.6 mg), and spinach ß-carotene (1.4 mg) to retinol were 2.0, 2.3, and 7.5 to 1 by weight, respectively. CONCLUSIONS: The ß-carotene in GR is as effective as pure ß-carotene in oil and better than that in spinach at providing vitamin A to children. A bowl of ~100 to 150 g cooked GR (50 g dry weight) can provide ~60% of the Chinese Recommended Nutrient Intake of vitamin A for 6-8-y-old children.

Analysis of the metabolites in apical area of Allium cepa roots by high resolution NMR spectroscopy method.

Elucidation of the molecular mechanisms determining the formation of various tissues and organs is one of the central problems of cell biology. High-resolution NMR spectroscopy was applied for the analysis of the metabolites produced at the various areas of the apical part of the onion Allium cepa roots. To this end, three samples were extracted from the root apex (the root cap, the meristem region and the cell elongation zone). These samples were noticeably different in the number of mitoses and the sets of metabolites. Furthermore, the complete stasis of the plant roots and tops growth was registered in heavy water. Comparison of the morphological and NMR data revealed their perfect agreement with the cellular processes occurring in the root apex. The root cap sample was characterized by the greatest mitotic activity reflected in the great variability of the chemical compounds extracted from this area, the high level of energy consumption, and the increased synthesis of the phosphocholines needed for the cell fission. Sample containing the cell elongation zone possessed the high sugar content, which is required for the cell-wall growth. Therefore, our data show that high-resolution NMR spectroscopy can be used for the identification of chemical compounds in the various regions of the onion root apical area.

Enhancement of skin permeation of high molecular compounds by a combination of microneedle pretreatment and iontophoresis.

A combination of microneedle pretreatment and iontophoresis was evaluated for the potential to increase skin permeation of drugs. Two model compounds with low and high molecular D(2)O and fluorescein isothiocyanate (FITC)-dextrans (FD-4, FD-10, FD-40, FD-70 and FD-2000; average molecular weight of 3.8, 10.1, 39.0, 71.2 and 200.0 kDa), respectively, were used and the effect of microneedle pretreatment and iontophoresis on their in vitro permeability was evaluated using excised hairless rat skin with a 2-chamber diffusion cell. Convective solvent flow through the skin was measured using a set of calibrated capillaries attached to the diffusion cell. The following results were obtained: (1) convective solvent flow (electroosmosis) during iontophoresis through microneedle-pretreated skin, 2.62+/-0.32 microL/cm(2)/h, was almost the same as through intact skin, 2.71+/-0.25 microL/cm(2)/h, and (2) the combination of microneedle pretreatment and subsequent iontophoresis significantly enhanced FD flux compared with microneedle pretreatment alone or iontophoresis alone, whereas no synergistic effect was found on the flux of D(2)O. These results suggest that the combination of iontophoresis with microneedle pretreatment may be a useful means to increase skin permeation of high molecular compounds.