Maynard M. Cohen

P‐31 Nuclear Magnetic Resonance Analysis of Brain: The Perchloric Acid Extract Spectrum

Abstract: Perchloric acid (PCA) extracts were prepared from liquid‐N2‐frozen guinea pig brains and their organophosphate profiles examined by P‐31 nuclear magnetic resonance (NMR) spectroscopy. Thirty‐two phosphorus‐containing brain metabolites were characterized and quantitated. A distinctive feature of brain tissue metabolism relative to that of other tissues probed by P‐31 NMR is its pronounced ribose 5‐phosphate content. Comparison of brain metabolite levels following control or sublethal cyanide treatment (4 mg/kg) revealed specific cyanide‐induced changes in brain metabolism. Brains from cyanidetreated animals were characterized by a reduced phosphocreatine content and elevated α‐glycerolphosphate and inorganic orthophosphate contents relative to control. P‐31 NMR spectra of brain PCA extracts at pH 7.2 were also obtained under conditions that approximate those used for in vivo and intact tissue in vitro P‐31 spectroscopic analyses. The spectra reveal nine separate resonance bands corresponding to: sugar phosphates, principally ribose 5‐phosphate (3.7δ); inorganic orthophosphate (2.2δ); glycerol 3‐phosphorylethanolamine (0.3δ); glycerol 3‐phosphorylcholine (−0.1δ); phosphocreatine (−3.2δ); adenosine tri‐(β‐ATP) and di‐(β‐ADP) phosphate ionized end‐groups (−6.2δ); α‐ATP, α‐ADP, and nicotinamide adenine dinucleotides esterified end‐groups (−11.1δ); uridine diphosphohexose, hexose esterified end‐groups (−13.0δ); and β‐ATP ionized middle group (−21.6δ). Knowledge of the phosphatic molecules that contribute resonances to the brain P‐31 NMR spectrum as well as understanding their magnetic resonance properties is essential for the interpretation of in vivo brain spectroscopic data as well as brain extract data, since these same compounds contribute to the intact brain P‐31 spectrum.

Acid soluble phosphates in the developing rabbit brain.

AN adequate source of energy is a prerequisite for the orderly maturation of the brain. Since the energy for developmental processes is provided principally through high energy phosphates, information concerning the development of these compounds is of central importance to the understanding of cerebral functional and structural differentiation. Previous reports of levels of high energy phosphates in the developing brain have been sparseandcontradictory and could not be correlated withmorphological and physiological differentiation. This investigation was undertaken to clarify the relationship of these compounds to the maturation of cerebral structure and function. Orthophosphate, phosphoethanolamine, creatine, and ascorbic acid were investigated i n addition to adenosine triphosphate and phosphocreatine because of their relationship to phosphorylation and development.

The effect of anoxia on the chemistry and morphology of cerebral cortex slices in vitro.

THE ULTIMATE aim in investigations of basic cerebral processes is the elucidation of mechanisms of the living brain in situ. However, all information necessary to the understanding of cerebral function cannot be obtained in the intact animal. In citro techniques have been substituted to delineate metabolic processes of short duratioii and to isolate individual reactions within more complex chemical cycles. The study of morphological response to physiological and pathological stimuli has been limited similarly, although less severely, by the necessity of removal and fixation of tissue after many of the structural changes are complete. As a consequence, it has been difficult to correlate physical structure and chemical state and results have been subject to erroneous interpretation. In previous investigations, a method was evolved whereby the morphology of cerebral slices metabolizing in v i m could be correlated with biochemical findings (COHEN, 1960). The present report is concerned with morphological changes resulting from anoxia in ritro and their biochemical counterparts. Cortical neurons were found to present characteristic histological alterations, and a metabolic site most sensitive to oxygen lack has been identified.

31P Nuclear Magnetic Resonance Studies of Anoxia in Aged Rat Brain

In addition to confirming previously reported findings related to changes in high-energy phosphates consequent to anoxia, 31P nuclear magnetic resonance studies of rat brain indicated that maintenance of energy-rich phosphates is less effective in the aged brain than in the mature cerebrum.

INTERRELATIONSHIPS OF GLUCOSE AND GLUTAMIC ACID METABOLISM IN DEVELOPING RABBIT BRAIN

CLINICAL and laboratory experience strongly suggests that the neonatal brain differs from the mature brain in its metabolic processes. During the neonatal period the concentrations of various chemical components of brain differ from those in adult tissue and are rapidly changing. (KREBS, EGGLESTON and HEMS, 1949; ROBERTS, PINCKNEY and FRANKEL, 1951; HIMWICH, PETERSEN and ALLEN, 1957; TOWER (review), 1959; COHEN and LIN, 1962). GAD? has been demonstrated to increase in activity as the brain matures (HIMWICH, PETERSEN and GRAVES, 1961). Study of respiration of brain slices obtained from animals shortly after birth conceivably could yield information to demonstrate differences in glutamate metabolism accompanying GAD changes. Information concerning glutamate metabolism in the very young animal, aside from concentration studies, is sparse indeed. Previous investigations of adult rat brain cortex in vitro demonstrated that glucose oxidation is depressed in the presence of glutamic acid, while glutamate oxidation appears to be enhanced by the presence of glucose (CHAIN, COHEN and POCCHIARI, 1962). In excess of 50 per cent of alpha amino nitrogen of brain is contributed by glutamic acid and its relatedcompounds, glutamine and GABA(TowER, 1959). These substances increase rapidly to the mature level in absolute concentration as the animal ages (TOWER, 1959). There is little variation in the absolute concentration of these cerebral components from one mammal to another. In the mature mammal, the average concentration of glutamic acid is about 11 micromoles/gram wet brain tissue, glutamine 4 micromoles/gram wet brain tissue, and GABA 2 micromoles/gram wet brain tissue (BERL and WAELSCH, 1958). Glutamic acid and GABA are almost exclusively associated with the neuronal fraction of cerebral tissue (TOWER, 1959; TSUKADA et al., 1960). As an example of the changes that take place with maturation, the following changes in concentration of glutamic acid in the rat take place: 1 day, 4.3 pmoles; 10 days, 6-4 pmoles; and 6 months, 10 pmoleslg wet brain tissue (HIMWICH, PETERSEN and ALLEN, 1957). HIMWICH, PETERSEN and GRAVES (1961), employing the technique of ROBERTS, YOUNG and FRANKEL (1951), assayed glutamic acid decarboxylase activity in the foetal and post-natal rabbit and noted moderate activity 15 days before birth, reaching a low rate of activity just before birth and then gradually increasing to adult rates

THE EFFECT OF GLUTAMIC ACID ON PHOSPHORUS METABOLISM IN CEREBRAL TISSUE PREPARATIONS

THE oxidation of glutamate by cerebral mitochondrial preparations has been reported to be efficiently coupled to phosphorylation (BRODY and BAIN, 1952; MCKHANN and TOWER, 1959). However, when cerebral cortex slices were employed, glutamate as substrate did not support steady-state concentrations of phosphocreatine (PC)

EFFECT OF GLUTAMIC ACID ON PHOSPHORYLATIVE ACTIVITY IN CEREBRAL TISSUE IN VITRO

comparable to those obtained with glucose (MCILWAIN, 1952). The possibility that increased potassium ion transport may play a role in this phenomenon was suggested by the studies of TERNER, ECGLESTON and KREBS (1950). Demonstration that addition of glutamate to the incubation medium did not produce an increase in intracellular potassium (PAPPIUS and ELLIOTT, 1956) made this possibility unlikely. Increased formation of glutamine from glutamate has been suggested as responsible for the lowered concentration of ATP (Acs, BALAZS and STRAUB, 1953) as well as PC (WOODMAN and MCILWAIN, 1961). The present studies were undertaken to re-examine this problem with techniques capable of determining the effect of glutamate on cerebral mitochondria1 as well as cortex slice metabolism, in the presence of glucose. The effect of glutamate on glycolysis and on the turnover of ATP was of special interest.

INFLUENCE OF CREATINE AND ETHANOLAMINE ON THE PHOSPHATE METABOLISM OF RABBIT CEREBRAL CORTEX SLICES IN VITRO

Although glutamate is unable to maintain concentrations of phosphocreatine in cerebral cortex slices it has been reported to support active phosphorylation in mitochondria1 preparations. This report is concerned with further investigation of this apparent discrepancy. I n cortical slice studies glucose supported concentrations of phosphocreatine of 1.40 and of ATP of 0 . 7 8 pmoleslg. The relative specific activity (R.S.A.) of ATP (S.A. tissue inorganic phosphate = 1) approximated 1, and of phosphoethanolamine 0.0s . All these values were markedly lower when glutamate alone was employed as substrate and approximated to those obtained in the absence of any added substrate. When glutamate in equimolar concentrations was added to a medium containing a glucose substrate, phosphocreatine concentrations fell to 0.84 pmoles/g. The decrease in the R.S.A. of ATP to 0 . 6 9 with glutamate as substrate would suggest decreased phosphorylation of ADP to ATP rather than an increased utilization (e.g. in the synthesis of glutamine) as has been suggested. In a glucose medium 27 mM K+ did not produce as low concentrations of phosphocreatine as did media containing 6 . 3 mM K+ with glutamate

Interrelationships of Glucose, Glutamate and Aspartate Metabolism in Developing Rabbit Brain

The use of cortical slices in the study of cerebral metabolism permits a wide range of experimentation, and yields information not readily obtainable from in situ preparations. The incubation media principally employed for such in vitro systems have been variations of the Krebs‐Ringer solutions (Krebs and Henseleit, 1932; Mcilwain,1959a; Heald, 1960). Although capable of supporting adequate respiration of the separated tissue, such media do not sustain all metabolic responses comparable to in vivo conditions (Mcilwain, 1959b). This paper is concerned with the effects of added creatine and ethanolamine on the metabolism of their phosphates in rabbit cerebral cortex slices in vitro. These compounds were chosen because of the sensitivity of phosphoryl creatine to metabolic deficiency as well as its important involvement in energy metabolism, and because of the rather wide‐spread presence of phosphoryl ethanolamine in animal tissues (Shaw, 1955; Long, 1961; Cohen and Lin, 1962). Some experiments were also done with slices from very young rabbits for comparative purposes.

Recovery of viable virus from poliomyelitis vaccine by use of monkeys pretreated with cortisone and x-radiation.

Publisher Summary Studies in this laboratory have demonstrated that glutamate and aspartate have different metabolic effects than glucose, when utilized as substrate for the in vitro metabolism of cerebral cortex slices prepared from mature animals. Clinical observations of newborn infants have revealed apparent normal central nervous system activity despite exceedingly low blood glucose concentration. The distinct possibility arises that glucose plays a different and less pivotal role in immature brain metabolism. This chapter is undertaken to assess the relative contribution of glucose, glutamate, and aspartate to oxidative decarboxylation in immature rabbit brain. Various combinations of U- 14 C labeled and non-radioactive glucose, L-glutamate, and L-aspartate are utilized as oxidizable substrates of immature rabbit brain slices. Glutamate depresses glucose oxidation beginning with the youngest brain studied. Aspartate depresses glucose oxidation in the 16-day-old animal. Glutamate is more readily oxidized than glucose when both are present in the media. This occurs at all ages studied here. The relative specific activity of CO 2 collected when U- 14 C labeled aspartate or U- 14 C labeled glucose are utilized as sole oxidizable substrates is comparable in the most immature animals. However, aspartate alone poorly supports respiration of the slices.