The Inorganic Phosphate and Phosphocreatine of Brain

As phosphate derivatives form such important intermediates between the functioning of tissues and the metabolism which supports function, we decided to investigate the levels of certain phosphates in brain slices before and during metabolism in vitro. Investigation of this type appeared all the more necessary because there is now an extremely interesting group of observations on the levels of phosphorus derivatives in the brains of experimental animals, before and after they have been subjected to various treatments. (For references and an assessment see Mcllwain, 1950; some results are quoted in Table 3 of the present paper.) The substances which have been determined following in vivo treatment of animals have included inorganic phosphate, creatine phosphate, and adenosine phosphates; and the treatments during which marked changes in these have been observed have included narcosis and induced convulsions. During these processes, the phosphate fractions in which greatest change has been observed have been inorganic and creatine phosphates, and we have accordingly commenced our studies by examining these compounds. In contrast to the data available from experiments in vivo, relatively little information is available of the concentrations of phosphates during in vitro metabolism of, for example, brain slices, though Macfarlane & Weil-Malherbe (1941) studied some ofthese compounds, especially during anaerobic glycolysis, in investigating the course of glycolysis. This deficiency is probably due to the extremely rapid changes which have been reported to take place in these substances, following death or injury to the head. We have fully confirmed the rapidity and magnitude of such changes, but have also found that when brain slices are prepared and maintained under conditions normally regarded as satisfactory for metabolic studies in vitro, the changes in phosphates in many cases ceased and could be reversed. Relatively stable levels in inorganic and creatine phosphates were found, which were to some extent characteristic of metabolic conditions, and which could approach those normal to the tissue in vivo. This study includes a subsidiary investigation into procedures for determining inorganic or creatine phosphates in small quantities oftissue. Two methods have been used. The first was an application of the well established calcium-ethanol separations, but determining, as well as phosphates, the creatine of creatine phosphate, precipitated at pH 8-2-8-5 by calcium salts in 80% ethanol. The second depended on the determination of inorganic phosphate under both Fiske & Subbarow's (1925, 1929) conditions and under those of Lowry & Lopez (1946). The difference between the two values gave the phosphates with the lability of phosphocreatine. Because of its convenience for metabolic studies we have largely employed guinea pig brain cortex during the present work. We have also determined the levels of phosphates in vivo in this species by the same methods as employed for the levels in vitro.