In this report we describe the development of a method for the concurrent reversed-phase high-performance liquid chromatographic separation and photodiode array detection of human plasma retinol, tocopherols and carotenes. For a single sample injection, retinol, retinyl acetate, α- tocopherol, γ-tocopherol, α-tocopheryl acetate, all-trans-α-carotene and all-trans-β-carotene, as well as the mono-cis geometrical isomers of β- carotene were separated and detected. Analytical separations were performed at a subambient temperature (0°C) over a Suplex pKb-100 reversed-phase analytical column with an isocratic mobile phase of methanol-methyl tert.- butyl ether-water (80:20:5, v/v/v) at a flow-rate of 0.8 ml/min for 60 min. Standards and samples were reconstituted in ethanol, and typically, 50 μl was injected for analysis. By HPLC, compounds of interest were clearly resolved and detectable at the picomole level.

The alga Dunaliella salina possesses the unique ability to accumulate up to 14 % of it's dry weight as β-carotene in response to stress conditions. This hyper-accumulation of β-carotene has led to the commercial exploitation of this alga for the biotechnological production of this important carotenoid. In order to maximise β-carotene production, a dual-stage process which separates the distinctive growth phase from the β-carotene accumulating stress phase has recently been patented. Preliminary laboratory studies showed that although stress factors such as high salinity and nutrient limitation enhance β-carotene accumulation in D. salina (± 10 pg.cell⁻¹), high light intensity was the single most important factor contributing to the induction of β-carotene accumulation in this alga (± 20 pg.cell⁻¹). Moreover, it was demonstrated that β-carotene accumulation can be further stimulated by exposing the alga to a combination of high light intensity, salt and nutrient stresses (± 30-60 pg.cell⁻¹). The response of D. salina to stress was shown to occur in two phases. The first phase occurred within 24 hours and was characterized most importantly by higher rates of β-carotene accumulation for all the stresses investigated. In cells exposed to multiple stress factors in mass culture, the β-carotene accumulation rate was as much as 9.5 pg.cell⁻¹.day⁻¹ in the first phase compared to only 3 pg.cell·day⁻¹ in the second phase. Since the rate of β-carotene accumulation was higher within the first 24 hours after exposure to stress, the first phase was considered crucial for stress-induced β-carotene accumulation. Characterization of this phase revealed that the stress response was multifaceted. Transition of cells from the growth stage to stress conditions was characterized by the following: 1) Change in cell volume. Hypersalinity caused cell shrinkage while cells exposed to nutrient limitation and/or high light intensity caused cells to swell. Restoration of cell volume was shown to occur within 8 hours for all stresses investigated. 2) Altered photosynthesis. Inhibition of both carbon fixation and oxygen evolution was demonstrated in cells immediately after exposure to multiple stress factors. 3) Production of abscisic acid. Intracellular ABA levels increased within 6-8 hours after exposure to all stresses investigated. The rise in intracellular ABA levels coincided with an increase or return to starting cell volume. High intracellular ABA levels were however transient and within 24 hours ABA began to partition into the culture medium. 4) Change in pigment composition. Changes in xanthophyll cycle pigment content was demonstrated soon after exposure to stress conditions. In hypersalinity shocked cells, initial epoxidation of zeaxanthin to violaxanthin and subsequent de-epoxidation to zeaxanthin occurred, whereas exposure to high stress resulted in an immediate and continued decrease in the epoxidation state indicating accumulation of zeaxanthin. A rapid rate of chlorophyll depletion was also demonstrated. In addition to the above responses a rapid decrease in growth rate during this phase was also noted. An interrelationship between cell volume change, ABA production, maintenance of xanthophyll cycle operation and β-carotene accumulation therefore appeared to exist. ABA production was shown to be stoichiometrically related to changes in xanthophyll content with r² = 0.84 and slope of the curve = 0.91 being achieved for high light stressed cells. This study therefore presents strong circumstantial evidence in support of a carotenoid origin for ABA in Dunaliella. In addition, enhanced β-carotene content was achieved by the application of exogenous ABA and related compounds suggesting a role for ABA as a regulator of the overall stress response. Furthermore, zeaxanthin accumulation was shown to be positively correlated ( r²≥ 0.81) to β-carotene accumulation for all the stresses investigated. The second phase was characterized by a return to homoeostasis of the physiological processes mentioned above, indicating acclimation of the cell to prevailing conditions. This stage was characterised by continued β-carotene accumulation and a decreased epoxidation state of the xanthophyll cycle which together appeared to sustain photosynthesis, allowing this organism to tolerate stress conditions.

The alga Dunaliella salina accumulates large quantities of β-carotene in response to certain environmental and physiological stresses. This hyper-accumulation process has been commercially exploited. However, the currently employed averaging or single-stage process produces β-carotene yields well below the genetic potential of the organism due to the inverse relationship between growth and secondary metabolite production. A dual-stage process, which separates the distinctive growth and secondary metabolite production stages of the alga, has been proposed. The broad aim of the research programme was to evaluate the practicality, scale-up and economic viability of a dual-stage β-carotene production process from D. salina. Preliminary laboratory studies showed that although stress factors such as high salinity and a range of nutrient limitations enhance β-carotene accumulation in D. salina, high light intensity is the single most important factor inducing β-carotene hyper-accumulation in the alga. Furthermore, the preliminary studies indicated that 6-carotene production could be successfully manipulated by the imposition of stress. The stress response of D. salina to high light stress was examined at a fundamental level. The relative partitioning of β-carotene between thylakoid membrane and interthylakoid globular β-carotene has revealed two responses to high light stress. The first is a response in which the alga adapts to the photoinhibitory effects of high light stress by the rapid accumulation and the peripheral localisation of Jl-carotene to the outer extremities of the chloroplast. This is followed by a maintenance response which is characterised by the recovery of the photosynthetic rate and cell growth. A possible interrelationship between the extent of the photo inhibitory response and the amount of β-carotene hyper-accumulation has been noted. An outdoor evaluation of the growth stage of the dual-stage system has demonstrated that D. salina can be grown in a relatively low salinity, nutrient sufficient medium for extended periods without overgrowth by small non-carotenogenic Dunaliella species. In addition, biomass productivities of three times greater than those obtained in the currently employed averaging system were achieved. The role of high light intensity in β-carotene hyper-accumulation was confirmed in outdoor scale-up stress pond studies. The studies demonstrated the feasibility of stress induced ll-carotene production in outdoor cultures of D. salina and β-carotene yields three times greater than those obtained in the currently employed averaging process were achieved. The dual-stage process imposes the specific requirement of viable cell separation on the harvesting system employed. A flocculation-flotation process and an air-displacement crossflow ultrafiltration system were developed and successfully evaluated for the separation of D. salina from the brine solution in a viable form. The extraction of β-carotene from D. salina was evaluated. Supercritical fluid extraction studies showed that the use of a co-solvent mixture of carbon dioxide and propane could effectively reduce the high extraction pressures associated with supercritical carbon dioxide extraction. In addition, a novel hydrophobic membrane assisted hot oil extraction process was developed which separates the complex oil-water emulsions produced during hot oil extraction of 6-carotene from wet D. salina biomass. Process design and economic evaluation studies were undertaken and showed that the economics of the dual-stage process offer significant advantages over the currently employed averaging process.

Call number: LD2668 .T4 1962 D43

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Thesis (Ph. D.)--Michigan State University. Dept. of Chemistry, 2006. / Title from PDF t.p. (viewed on Nov. 20, 2008) Includes bibliographical references. Also issued in print.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1952. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 53-56).

The object of this study is to determine the intake (in food) and output (in feces) of vitamin A and carotene of several groups of college women living in the Home Management House.