Labeling of glutamate, glutamine, and aspartate in brain from c14 aspartate and acetate / W. J. Ncklas D. D. Clarke and S. Berl Chemistry Dept., Fordham University and Col. of Physicians and Surgeons, Columbia University

Guinea pig brain cortex slices incubated in media containing U-C14 aspartic acid form glutamine of higher specific activity than glutamic acid (Fed. Proc. -25, 713 (1966) ). Glutamic acid and glutamine from such experiments were enzymatically decarboxylated and the o/o of label in C-1 ranged from 37 to 42o/o. These values exceed the maximum predicted from the known operation of the citric acid cycle viz. 33o/o. When l-C14 acetate was injected into mice or incubated with brain slices the aspartate isolated from brain had more than 50o/o of the label in C-4. From the known operation of the citric acid cycle it would be predicted that C -1 and C-4 should be equally labeled. The results of both series of experiments can be explained if it is assumed that the pyruvate moiety of oxaloacetate exchanges with unlabeled pyruvate from carbohydrate metabolism more readily than does the beta carboxyl group with COz. Supported by grants from NINDB and NIGMS

Effect of fluoroacetate on amino acid metabolism / W. J. Nicklas, D. D. Clarke, and S. Berl. Chem. Dept., Fordham Univ., and Col. of Physicians and Surgeons, Columbia Univ., New York, N. Y.

Guinea pig brain cortex slices incubated in media containing U-14-aspartate or glutamate (GA) (J. Neurochem. in press) form glutamine ( GM) of relative specific activity (RSA)\u3e 1 (GA=l). In the presence of 10-3 fluoroacetate (FA) the RSA of GM decreased to values considerably \u3c 1 without any changes in levels of amjno acids; the % of label in GA doubled whilA that in GM decreased correspondingly. Similar results were obtained with 1-14 C-acetate as tracer in vivo (mice) and in vitro. RSA\u27 s of Grvl \u3e 1 have been explained by the compartmentation of GA in cerebral cortex. Consequently the effect of GA may be explained by an inhibition of the flow of metabolites through that pool of GA used preferentially for GM synthesis. This is consistent with the concept of the existence, in brain, of more than one citric acid cycle, one of which is capable of activating acetate much more readily than the other(s) and hence more subject to inhibition by FA. Supported by grants from NINDB and the Cerebral Palsy Foundatio

Decarboxylation studies of glutamate, glutamine, and aspartate from brain labelled with [I-14]acetate, L-[U-14C]-aspartate, and L-[U-14C]glutamate / W.J. Nickls and D. D. Clarke Chemistry Department, Fordham University, Bronx, N.Y. and S. Berl Department of Neurology, College of Physicians and Surgeons, Columbia University, New York, N.Y.

Studies in vivo and in vitro of the distribution of label in C-1 of glutamate and glutamine and C-4 of aspartate in the free amino acids of brain were carried out. [1-14C]Acetate was used both in vivo and in vitro and L-[U-14C]aspartate and L-[U-14C]glutamate were used in vitro. (1) The results obtained with labelled acetate and aspartate suggest that CO2 and a 3-carbon acid may exchange at different rates on a CO2-fixing enzyme. (2) The apparent cycling times of both glutamate and glutamine show fast components measured in minutes and slow components measured in hours. (3) With [l-14C]acetate in vitro glutamine is more rapidly labelled in C-1 than is glutamate at early time points; the curves cross over at about 7 min. (4) The results support and extend the concept of metabolic compartmentation of amino acid metabolisn1 in brai

Interaction of catecholamine and amino acid metabolism in brain: effect of pargyline and l-dopa / W. J. Nicklas, S. Berl, and D. D. Clarke Department of Neurology, College of Physicians and Surgeons, Columbia University, 640 West 168th Street, New York, NY 10032 and Department of Chemistry, Fordham University, Bronx, NY 10458, U.S.A.

The combination of L-DOPA and pargyline caused a decrease in level of aspartate and an increase in that of glutamine in vivo in cerebral cortex, cerebellum, brain stem, hypothalamus, neostriatum and cervical cord of rat. There was also a decreased incorporation of radioactivity from [1-14C]acetate into amino acids in rico, most notably in cerebellum and brain stem. The labelling of glutamine was especially affected. In addition, cortical slices were prepared from guinea pigs which had been pretreated with pargyline. These slices were incubated with and without 1 mM L-DOPA in media containing [l- 14C]acetate. Pargyline alone caused a stimulation of the labelling of glutamate and aspartate but not glutamine and GABA ; the levels of aspartate and GABA were greater than in control slices. The addition ofL-DOPA to slices from pargylinized animals caused a severe decrease in glutamine labelling but not in that of glutamate or aspartate; the level of glutamine was increased while that of glutamate was decreased. The results are discussed in terms of the known biochemical and morphological compartmentation of amino acids in brain. It is suggested that catecholamines, in the process of functioning as transmitters, may also function as metabolic regulators of other transmitters, e.g. amino acids, as well as of the energy required for balanced neuronal functio

Compartmentation of glutamic acid metabolism in brain slices / S. Berl, W.J. Nicklas, and D.D. Clarke Department of Neurology, College of Physicians and Surgeons, Columbia University, Department of Chemistry, Fordham University, New York

(1) Compartmentation of glutamate metabolism in brain cortex previously observed only in vivo, has now been demonstrated in vitro. This was shown by using [U-14C]aspartate and [U-14C]glutamate as tracer substrates. (2) Preparation and maintenance of the slices at 0° resulted in reversible inhibition of glutamine synthesis. Preincubation at 37° for 10 min or preparation of the slices at room temperature partially overcame this inhibition. (3) Transfer to fresh medium after preincubation had an added stimulatory effect on glutamine synthesis. (4) Incubation in high K+ medium (27 mM) altered the relative specific activity of glutamine. (5) The data are in keeping with the postulate of the existence of at least two different pools of citric acid cycle intermediates in the cerebral corte

Oxidative metabolism of 4-aminobutyrate by rat brain mitochondria: inhibition by branched-chain fatty acid / J. Cunningham, D. D. Clarke, and W.J. Nicklas

The oxidation of 4-aminobutyric acid (GABA) by nonsynaptosomal mitochondria isolated from rat forebrain and the inhibition of this metabolism by the branched-chain fatty acids 2-methyl-2-ethyl caproate (MEC) and 2,2- dimethyl valerate (DMV) were studied. The rate of GABA oxidation, as measured by 02 uptake, was determined in medium containing either 5 or 100 mM-[K+]. The apparent Km for GABA was 1.16 ± 0.19 mM and the Vmax in state 3 was 23.8 + 5.5 ng-atoms O2 ·min-1.mg protein-1 in 5 mM-[K+]. In a medium with 100 mM-[K+] the apparent K111 was 1.11 + 0.17 mM and V max was 47.4 + 5. 7 ng-atoms 0 2 · min-1. mg protein-1 • The Ki for MEC was determined to be 0.58 + 0.24 or 0.32 + 0.08 mM, in 5 or 100 mM-[K+], respectively. For DMV, the Ki was 0.28 + 0.05 or 0.34 + 0.06 mM, in 5 or 100 mM-[K+] medium, respectively. The O2 uptake of the mitochondria in the presence of GABA was coupled to the formation of glutamate and aspartate ; the ratio of oxygen uptake to the rate, of amino acid formation was close to the theoretical value of 3. Neither the [K+] nor any of the above inhibitors had any effect on this ratio. The metabolism of exogenous succinic semialdehyde (SSA) by these same mitochondria was also examined. The Vmax for utilization of oxygen in the presence of SSA was much greater than that found with exogenously added GABA, indicating that the capacity for GABA oxidation by these mitochondria is not limited by SSA dehydrogenase. In addition, the branched-chain fatty acids did not inhibit the metabolism of exogenously added SSA. Thus, the inhibitors examined apparently act by competitively inhibiting the GABA transaminase system of the mitochondri

Glial cells and metabolic compartmentation / S. Berl, W.J. Nicklas, and D.D. Clarke Dept. of Neurology, MT. Sinai School of Medicine, N.Y., N.Y. 10029 Dept. of Chemistry, Fordham University, N.Y. 10458, U.S.A.

The study of metabolic compartmentation in brain has been under investigation for some twenty years. Most interpretations of the data have focused on attempts to assess the relative contributions of glia and neurons to the metabolic pictures since anatomically and functionally they are distinct. It is very probable that these cell types contain other metabolic compartments based on anatomic and subcellular subdivisions. However, the presently available experimental data is insufficient to specify precisely the total number of such metabolic pools functioning in nervous tissue. The initial observations that set in motion most of the work were a) in the adult animal there is a very marked blood-brain barrier to glutamate, and b) labelled glutamate administered intracranially is converted to glutamine much more rapidly than it mixes with the total glutamate endogenous to the brain. This results in a radiospecific activity of isolated glutamine several times that of its precursor glutamate even at time periods of a few minutes. This characteristic phenomenon could also be elicited with other labelled substrates e.g. aspartate as well as by peripheral administration of labelled substrates which more readily enter the brain from the circulation such as acetate, leucine, bicarbonate and ammonium salts. These in vivo studies were confirmed by in vitro studies which allow greater experimental manipulation. One of the ways of studying these compartments and determining their physiological importance is to perturb the normal metabolic state and attempt to relate the subsequent alterations in metabolic flux of labelled tracers to hypothesis of compartment localizatio

Psychometric evaluation of the Pittsburgh sleep quality index in cancer patients

Abstract: This report summarizes findings related to the psychometric properties (internal consistency and construct validity) of the Pittsburgh Sleep Quality Index (PSQI) and discusses issues related to its use based on data from two clinical studies with diverse samples of cancer patients. Subjects completed a questionnaire that included the PSQI, the Schwartz Cancer Fatigue Scale, and specific demographic, disease, and treatment variables. There were complete data on 170 (of 214) cases in Study 1 and 249 (of 259) cases in Study 2. The Cronbach's alpha for the Global Sleep Quality scale was 0.81 in Study 1 and 0.77 in Study 2. A comparison of Global Sleep Quality in two contrasting groups with low and high fatigue yielded statistically significant differences in both samples. Psychometric evaluation supports its internal consistency reliability and construct validity. However, the scoring is rather cumbersome and raises questions regarding level of measurement and appropriate analysis techniques