Improved conditions for the preservation and extraction of polyphosphoinositides

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Abstract

  • 1.

    1. Conditions have been determined which permit the recovery of greater amounts of di- and triphosphoinositides from tissues than previously obtained, especially when some time must be allowed to elapse before the extraction procedure can be started. Addition of CaCl2 at the optimal level of 60 μmoles/g of tissue to the chloroform-methanol (1:1, v/v) (at least 15 vol.) used for the extraction of the bulk of the lipids results in the subsequent isolation of larger quantities of polyphosphoinositides and especially diphosphoinositide. When tissue homogenates or subcellular fractions are to be analyzed, the use of an alkaline homogenization medium largely prevents the degradation of these lipids for several hours. Keeping the temperature near 0 °C is desirable.

  • 2.

    2. When these conditions were combined with the usual extraction of polyphosphoinositides with acidified solvents and separation by thin-layer chromatography, levels of the two lipids in brains of 1- and 7-day-old rats were similar at both ages, but diphosphoinositide was about 50 % higher than triphosphoinositide. During standing at room temperature levels in 7-day-old brains fell more rapidly than in 1-day-old brains.

  • 3.

    3. Levels in gray matter of 34-day-old rats were higher than previously found (Hauser G., Eichberg J. and Gonzalez-Sastre F. (1971) Biochim. Biophys. Acta 248, 87–95) but the marked disappearance during a period of anoxia was confirmed.

  • 4.

    4. Adult rat kidney values were increased much less than those for brain when the initial extraction was carried out in the presence of CaCl2.

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