Abstract— Mouse brain subcellular fractions were prepared at 1, 12, and 24 h and 3 and 8 days after intracerebral injections of [1‐14C]arachidonate. Initially, radioactivity was mainly distributed in the microsomal and synaptosomal fractions, but the proportion of radioactivity in the myelin increased from 5 to 16% within 8 days. Radioactivity of the microsomal lipids started to decline at 1 h after injection, and the decay was represented by two pools with half‐lives of 19 h and 10 days, respectively. Radioactivity in the synaptosomal and myelin fractions did not reach a maximum until 24 h after injections. The half‐life for turnover of synaptosomal lipids was 9 days. The decline of radioactivity measured in the microsomal fraction was due mainly to diacyl‐GPC and diacyl‐GPI, since radioactivity of other phosphoglycerides (diacyl‐GPS, diacyl‐GPE and alkenyl‐acyl‐GPE) continued to increase for 12‐24 h. In this fraction, half‐lives of 10‐14 h were obtained for the fast turnover pools of diacyl‐GPC and diacyl‐GPI, and slow turnover pools with half‐lives of 7 days for diacyl‐GPI and 10‐14 days for other phosphoglycerides were also present. Among the synaptosomal phosphoglycerides, radioactivity of diacyl‐GPI declined in a biphasic mode, thus exhibiting half‐lives of 5 h and 5 days. Incorporation of labelled arachidonate into diacyl‐GPE and diacyl‐GPS in the synaptosomal fractions was observed for a period of 24 h. The half‐lives for these phosphoglycerides ranged from 8 to 12 days. Results of the study have demonstrated the presence of small pools of arachidonoyl‐GPI in synaptosomal and microsomal fractions which were metabolically more active than other arachidonoyl containing phosphoglycerides.

The metabolism of arachidonate in brain membrane phosphoglycerides was investigated in vivo by intracerebral injection of labeled arachidonate and by in vitro assay of enzymic systems associated with the metabolism. After intracerebral injection, labeled arachidonate was incorporated rapidly into brain phosphoglycerides with radioactivity distributed mainly in diacyl‐sn‐glycero‐3‐phosphoinositols (GPI) and diacyl‐sn‐glycero‐3‐phosphocholines (GPC). Some evidence of a metabolic relationship between diacyl‐sn‐glycerophosphoinositols (diacyl‐GPI) and diacylglycerols was observed. Among the phosphoglycerides labeled with [14C] arachidonoyl groups, diacyl‐GPI were most rapidly metabolized in brain microsomal and synaptosomal fractions. The decay of diacyl‐GPI in brain synaptosomes may be represented by two pools with half‐lives of 5 hr and 5 days. Three types of enzymic systems related to metabolism of the polyunsaturated fatty acids in brain were investigated. The first system involves the cyclic events relating the ATP‐dependent activation of polyunsaturated fatty acids (PUFA) to their acylCoA by the acylCoA ligase and subsequent hydrolysis of acylCoA to free fatty acids by the acylCoA hydrolase. It is apparent that fatty acid activation and hydrolysis is under strigent control in order to maitain suitable levels of free fatty acids and acylCoA in the brain tissue for various metabolic use. Factors involved in the regulation may include the level of ATP, divalent cations and the nature of substrates. The second enzymic system pertains to deacylation via phospholipase A2 and reacylation via the acyltransferase of membrane phosphoglycerides. In brain tissue, activity of the acyl transferase is generally higher than that of the phospholipase A2. Factors known to affect specificity of the acyltransferase include substrate concentration and the nature of the acyl groups and lysophosphoglycerides. The acyltranferase(s) in brain preferentially transfers arachidonate to 1‐acyl‐GPI. Activity of the acyltransferase can be inhited by a number of lypophilic compounds including local anesthetics and cell surface agents. Activity of the phospholipase A2 in brain may depend on the physical form of the substrates, i.e., whether the substrates are in monomeric or micellar form. The third process is associated with the degradation of diacyl‐GPI by enzymes present in brain subcellular membranes. Incubation of brain subcellular membranes with 1‐acyl‐2‐[14C] arachidonoyl‐GPI yielded labeled diacylglycerols and arachidonate. The phospholipase C action is specific for hydrolysis of diacyl‐GPI. The arachidonate released from incubation of labeled diacyl‐GPI may be the result of phospholipase A2 action which is not specific for diacyl‐GPI or the hydrolysis by lipase acting on the diacylglycerols formed from the phospholipase C activity. Enzymic hydrolysis of diacyl‐GPI is most active in the microsomal fraction, but uoon disruption of synaptosomes, enzyme in synaptic plasma membranes is also active in degradating this glycerophospholipid. In general, the results of in vitro studies are in good agreement with those observed in vivo and the information yielded has contributed towards understanding the metabolism of polyunsaturated fatty acids in brain subcellular membranes.

A method is described for the quantification of the constituent O-alkyl and O-alk-1-enyl glycerols of neutral lipids or phospholipids. The method involves chemical degradation, the preparation of derivatives, and quantification by gas chromatography using internal standards. Alk-l-enyl moieties are converted to alkyl substituted dioxanes in the presence of 1,1-dimethoxyheptadecane as standard; alkyl glycerols are analyzed as isopropylidene derivatives using 1-O-heptadecyl glycerol as internal standard. The method is applied to the quantification of alkyl and alk-l-enyl glycerols derived from total lipids of rat heart, liver, testes, and brain and of various transplantable tumors, i.e. amelanotic melanoma, melanoma B16, sarcoma T241, and Novikoff hepatoma. The levels of alkyl glycerols range from 0.11-1.07% total lipids and those of alk-l-enyl glycerols from 0.49-5.03%. The data are compared to those obtained by other methods.

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