Lipids of nervous tissue: composition and metabolism.

We examined the metabolic disposition and brain distribution of an unsaturated fatty acid, [1-14C]arachidonate, between 5 and 240 min following its intravenous bolus injection in unanesthetized adult rats. Injected [1-14C]arachidonate was cleared rapidly from plasma, with less than 10% remaining by 2 min. Total brain radioactivity, 0.2% of the injected dose, was near maximal by 5 min, reached a peak by 15 min, then slowly declined. Radioactivity in brain lipids constituted greater than 82% of the total brain radioactivity at all times. Radioactivity in aqueous-soluble metabolites was greatest at 5 min (13% of total) and declined to 5% by 240 min. Protein pellet-associated radioactivity gradually rose to a peak of 7% by 120 min. Within the lipid fraction, more than 92% of radioactivity was in glycerolipids, with greater than 81% in phospholipids. Radioactivity in inositol phosphoglyceride was maximal at 5 min (47% of phospholipid radioactivity); and declined to 34% by 20 min, whereas radioactivity in choline phosphoglyceride peaked at 15 min (41% of phospholipid radioactivity) and was constant thereafter. In contrast, radioactivity in ethanolamine phosphoglycerides increased from 7 to 17% during the course of the experiment. Quantitative autoradiography of brain sections indicated incorporation of [1-14C]arachidonate into gray-matter regions was 1.5- to threefold that into white-matter regions. The data were analyzed in terms of a model for brain fatty acid uptake from plasma. Estimates of unidirectional transfer constants, k, for [1-14]arachidonate from plasma to brain regions with an intact blood-brain barrier ranged from 0.0005 to 0.0015 ml.sec-1.g-1 and were correlated with those for [9,10-3H]palmitate. The results indicate that brain phospholipid metabolism in awake animals can be examined regionally and quantitatively using intravenous injection of [1-14C]-arachidonate combined with quantitative autoradiography and biochemical analysis.

Intravenous injection of [1-14C]arachidonate to examine regional brain lipid metabolism in unanesthetized rats.

Radioactivity within individual brain compartments was determined from 5 min to 44 h after intravenous injection of [14C]palmitate into awake Fischer-344 rats, aged 21 days or 3 months. Total radioactivity peaked broadly between 15 min and 1 h after injection, declined rapidly between 1 and 2 h, and then more slowly. In 3-month-old rats, the lipid and protein brain fractions were maximally labeled within 15 min after [14C]palmitate injection, then retained approximately constant label for up to 2 days. Radioactivity in the aqueous brain fraction comprised mainly radioactive glutamate and glutamine, and peaked at 45 min, when it comprised 48% of total brain radioactivity, then decreased to 27% of the total at 4 h, 15% at 20 h, and 10% at 44 h. Percent distribution of radioactivity within the different brain compartments, 4 h after intravenous injection of [14C]palmitate, was similar in 21-day-old and 3-month-old rats, despite higher net brain uptake in the younger animals. The results indicate that about 50% of plasma [14C]palmitate that enters the brain of adult rats is incorporated rapidly into stable protein and lipid compartments. The remaining [14C]palmitate enters the aqueous fraction after beta-oxidation, and is slowly lost. At 4 h after injection, 73% of brain radioactivity is within the stable brain compartments; this fraction increases to 86% by 20 h.

Utilization of plasma fatty acid in rat brain: distribution of [14C]palmitate between oxidative and synthetic pathways.

— [1-14C]Arachidonic acid was incorporated into brain lipids with a half-life of approx. 5 min. Within 40 min after intra-cerebral injection, radioactivity was distributed mainly among the diacyl-sn-glycero-3-phosphorylcholine (45 per cent), diacyl-sn-glycero-3-phosphorylinositol (22 per cent), diacyl-sn-glycero-3-phosphorylethanolamine (14 per cent) and triacylglycerols (9 per cent). At comparable times, the proportions of radioactivity distributed in diacyl-sn-glycero-3-phosphorylserines and alkenylacyl-sn-glycero-3-phosphorylethanolamines were relatively small. Radioactivity was initially incorporated into the phosphatidio acids and diacylglycerols before labelling of the triacylglycerols and other phosphogly-cerides. The relative specific activity of diacylglycerols was maximum between 3–6 min after injection. Due to the small level of diacyl-sn-3-phosphorylinositol present in brain, its relative specific radioactivity was higher than other types of brain phosphoglycerides. Results of the experiment thus indicate that labelled arachidonic acid is an excellent precursor for metabolic studies with regard to acyl groups present in the 2-position of the phosphoglyceride molecules. Furthermore, this labelled precursor is specially useful in studies related to metabolism of diacyl-sn-glycero-3-phosphorylinositol in brain.

The metabolism of (1-14c)arachidonic acid in the neutral glycerides and phosphoglycerides of mouse brain.

—Phosphatidic acids of rat brain were shown to be predominantly of the monoenoic class while diacylglycerols and phosphatidylinositols were constituted mainly by tetraenes. Metabolic inter-relationships were examined after intraventricular injection of [14C]glycerol, [3H]arachidonate and [9,10-3H2]stearate. In each case, diacylglycerols were most highly labelled, although a small pool of arachidonate was located in brain triacylglycerols, mainly esterified to a primary carbinol, with extremely high turnover rate. Fractionation of the lipids showed a preferential synthesis of disaturated, monoenoic and polyenoic classes (>4 double bonds) of phosphatidic acid, diacylglycerol and phosphatidylinositol. The high flux of [3H]stearate through disaturated species of phosphatidic acid and diacylglycerol could be partially suppressed by simultaneous injections of unsaturated fatty acids, both probably consequences of perturbing the very small brain pool of free fatty acids. Kinetics of labelling of phosphatidylinositols were consistent with formation of arachidonoyl-containing species by acyl transfer mechanisms with disaturated and oligoenoic classes serving as precursors. Although the profile of molecular classes of diacylglycerol and phosphatidylinositol strongly suggests a metabolic relation, there was no obvious evidence for this in the kinetic studies of the whole brain lipids. Such relation, however, may have been masked by the rapid flow of radioactivity from phosphatidic acids to diacylglycerols.

Selective synthesis of molecular classes of phosphatidic acid, diacylglycerol and phosphatidylinositol in rat brain.

The incorporation of [1-14C]-oleic acid into the neutral glycerides and phosphoglycerides of adult mouse brain was examined between 1 and 80 min after intracerebral injection. Radioactivity of the free oleic acid in brain decreased rapidly with a half-life of ca. 5 min. The specific radioactivity of the phosphatidic acids was highest at 1 min after injection. This was followed by the diacylglycerols and triacylglycerols which attained a maximum specific radioactivity at 3 and 20 min after injection, respectively. Specific radioactivities of the neutral glycerides were higher than the phosphoglycerides. A larger proportion of the radioactivity in the diacylglycerols was transferred to the phosphoglycerides than to the triacylglycerols. Among the phosphoglycerides, radioactivity was actively incorporated into the inositol phosphoglycerides. The specific radioactivity of the inositol phosphoglycerides was higher than the diacylsn-glycero-3-phosphorylcholines, and the kinetics of incorporation of radioactivity into these lipids was also different. A water soluble material was found which showed maximum specific radioactivity at 6–10 min after injection. The properties of this water soluble material suggested that it may be an intermediate involved in the acyl group metabolism of phosphoglycerides in brain.

T. M. Yau

Papers

Incorporation of (1-14c)-oleic acid into neutral glycerides and phosphoglycerides of mouse brain.