A single-step method for rapid extraction of total lipids from green microalgae.

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Microalgae produce a wide range of lipid compounds of potential commercial interest. Total lipid extraction performed by conventional extraction methods, relying on the chloroform-methanol solvent system are too laborious and time consuming for screening large numbers of samples. In this study, three previous extraction methods devised by Folch et al. (1957), Bligh and Dyer (1959) and Selstam and Öquist (1985) were compared and a faster single-step procedure was developed for extraction of total lipids from green microalgae. In the single-step procedure, 8 ml of a 2∶1 chloroform-methanol (v/v) mixture was added to fresh or frozen microalgal paste or pulverized dry algal biomass contained in a glass centrifuge tube.

The biomass was manually suspended by vigorously shaking the tube for a few seconds and 2 ml of a 0.73% NaCl water solution was added. Phase separation was facilitated by 2 min of centrifugation at 350 g and the lower phase was recovered for analysis. An uncharacterized microalgal polyculture and the green microalgae Scenedesmus dimorphus, Selenastrum minutum, and Chlorella protothecoides were subjected to the different extraction methods and various techniques of biomass homogenization. The less labour intensive single-step procedure presented here allowed simultaneous recovery of total lipid extracts from multiple samples of Centrifuge tube green microalgae with quantitative yields and fatty acid profiles comparable to those of the previous methods. While the single-step procedure is highly correlated in lipid extractability (r² = 0.985) to the previous method of Folch et al. (1957), it allowed at least five times higher sample throughput.

Santowhite, a commercial antioxidant used in the manufacture of polypropylene, contaminates 12-ml polyallomer tubes to the extent of 0.2-0.3 mg/tube.

  • It is distributed through the plastic and appears as a microscopic dust on the tubes’ surfaces. In the preparation of polysomes from rat liver by standard methods, approximately 15% of the antioxidant in previously water-washed tubes was removed by suspension of the polysome pellet with stirring in 1-2 ml of Tris buffer, pH 7.8.
  • The polyallomer impurity has been shown to be identical with Santowhite, which is 4,4′-butylidene-bis(6-tert-butyl-m-cresol), by UV, mass, and NMR spectra.
  • It is not uniformly removed from polyallomer tubes by common detergents but is removed by washing with acetone, to which the plastic is resistant.
  • Colloidal silica–aluminum modified–PVP density gradient centrifugation: centrifuge tube wall cell adherence, aggregation, separation properties and comparison to BSA and Ficoll.

A method is described for the inexpensive and easy preparation of colloidal silica–aluminum modified–polyvinylpyrrolidone (CS-AM-PVP) density gradient centrifugation medium.

Using density gradient centrifugation, several cell separation and biochemical characteristics were studied: centrifuge tube wall cell adherence, mouse spleen cell density distribution, rebanding properties, mitogen response and cell aggregation. Cell adherence to the centrifuge tube wall using density gradients of CS-AM-PVP was compared with density gradients of bovine serum albumin and Ficoll.

Few cells adhered to the centrifuge tube wall when CS-AM-PVP was used as a gradient medium; whereas, significant cell adherence to the centrifuge tube cell wall occurred when bovine serum albumin or Ficoll was used as a gradient medium. The CS-AM-PVP gradient medium did not inhibit the response to mitogens of mouse spleen cells which had been separated into density subpopulations in a discontinuous CS-AM-PVP density gradient, caused a minimum amount of cell aggregation, and was found to be non-toxic.

Islet transplantation is a promising method for restoring normoglycemia and alleviating the long term complications of diabetes.

  1. Widespread application of islet transplantation is hindered by the limited supply of human islets and requires a large increase in the availability of suitable insulin secreting tissue as well as robust quality assessment methodologies that can ensure safety and in vivo efficacy.
  2. We explore the application of nuclear magnetic resonance (NMR) spectroscopy in two areas relevant to beta cell engineering and islet transplantation: (1) the effect of genetic alterations on glucose metabolism, and (2) quality assessment of islet preparations prior to transplantation.
  3. Results obtained utilizing a variety of NMR techniques demonstrate the following: (1) Transfection of Rat1 cells with the c-myc oncogene (which may be involved in cell proliferation and cell cycle regulation) and overexpression of Bcl-2 (which may protect cells from stresses such as hypoxia and exposure to cytokines) introduce a wide array of alterations in cellular biochemistry, including changes in anaerobic and oxidative glucose metabolism, as assessed by 13C and 31P NMR spectroscopy.
  4. Overnight incubation of islets and beta cells in the bottom of centrifuge tubes filled with medium at room temperature, as is sometimes done in islet transportation, exposes them to severe oxygen limitations that may cause cell damage. Such exposure, leading to reversible or irreversible damage, can be observed with NMR-detectable markers using conventional 13C and 31P NMR spectroscopy of extracts.
  5. In addition, markers of irreversible damage (as well as markers of hypoxia) can be detected and quantified without cell extraction using high-resolution magic angle spinning 1H NMR spectroscopy. Finally, acute ischemia in a bed of perfused beta cells leads to completely reversible changes that can be followed in real time with 31P NMR spectroscopy

Plasma membranes were isolated from rat pheochromocytoma cells (PC-12) grown in spinner culture.

The rapid and simple isolation procedure consisted of a differential and isopycnic centrifugation (in a linear sucrose gradient) with the aid of a high capacity fixed angle rotor equipped with siliconized centrifuge tubes. The isolated membranes were closed and osmotically active vesicles (about 0.3 micron in diameter) with a mean intravesicular water space of 1.84 microliters/mg protein. In the presence of an inward gradient of sodium chloride and an outward gradient of potassium, [3H]noradrenaline (50 nM) was taken up and accumulated 550-fold (at 31 degrees C).

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The uptake and accumulation of [3H]noradrenaline was temperature-sensitive and inhibited by the tricyclic antidepressant desipramine. Membrane vesicles isolated from PC-12 cells represent a useful model for the investigation of the molecular mechanism of the neuronal noradrenaline transport system.

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