Biochimica et Biophysics Acta, 326 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGF (1973) 201-209 Q Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 56341 IMPROVED CONDITIONS FOR THE PRESERVATION AND EXTRACTION OF POLYPHOSPHOINOSITIDES GEORGE HAUSER and JOSEPH EICHBERG Biological Research Laboratory, McLean Hospital, Belmont, Mass. 02 I 78 and Department of Biological Chemistry, Harvard Medical School, Boston, Mass. 02 IIT (U.S.A.) (Received June sth, 1973) SUMMARY I. Conditions have been determined which permit the recovery of greater amounts of di- and triphosphoinositides from tissues than previously obtained, es- pecially when some time must be allowed to elapse before the extraction procedure can be started. Addition of CaCl, at the optimal level of 60 pmoles/g of tissue to the chloroform-methanol (I : I, 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 polyphospho- inositides 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 o “C is desirable. 2. When these conditions were combined with the usual extraction ofpolyphos- phoinositides with acidified solvents and separation by thin-layer chromatography, levels of the two lipids in brains of I- 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 r-day-old brains. 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. Adult rat kidney values were increased much less than those for brain when the initial extraction was carried out in the presence of CaCl,. INTRODUCTION Studies of the distribution and metabolism of brain polyphosphoinositides have been hampered by the limitation that the levels of both di- and triphosphoinositides fall rapidly after death 1-Z Prevention of this phenomenon requires either removal sf . the brain after immediate freezing of the head in liquid N, or prompt dissection and freezing of tissue samples. Consequently, although the preferential deposition of polyphosphoinositides in myelin has been thoroughly documented3-‘, it has been