Effect of Seizure Induced by Hydroxymethylpyrimidine on the Synthesis of Rat Brain Amino Acids from Glucose-U-14C1

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1 THE JOURNAL OF VITAMINOLOGY 15, (1969) Effect of Seizure Induced by Hydroxymethylpyrimidine on the Synthesis of Rat Brain Amino Acids from Glucose-U-14C1 TEIzo FUKUSHIMA AND YoSHITSUGU NOSE 2 Biochemical Institute, Kyoto Pref ectural University of Medicine, Kawar-arnachi, Kyoto (Post No. 602) (Received January 20, 1969) The free amino acids and glucose of rat brains were determined after separation by high-voltage paper electrophoresis and the isotopic incorporation from glucose-u-14c into the individual amino acids was measured at the preconvulsive stage induced by hydroxy methylpyrimidine. The absolute level of each amino acid was not so decisively varied, but the incorporation of labeled 14C-glucose into the amino acids was considerably lowered in the drug-treated group. The change of incorporation among each amino acid was relatively constant. It is assumed that the metabolic disturbances of glucose were widely brought about in the drug-treated rats. These changes were not restored by the concomitant admin istration of pyridoxal-hcl, though the convulsion was completely protected. The running fit incited by OMPm injected to rats and some other animals could be overcome by the structurally related compound, pyridoxine (1, 2). Several investigations of the seizure caused by not only OMPm, but also the. so-called toxopyrimidine group compounds have been carried out to elucidate the convulsion mechanism (3-9). Some enzymatic evidences for the antagonism between vitamin B6 and toxopyrimidine have been obtained with in vitro experiments (10-14), but the changes in the amino acid levels of rat brain induced by these compounds are still under scrutiny (15, 16), and the recovery of the amino acids of rat brain in the protected state from convulsion with vitamin B6 remains obscure. In the present paper, we report the levels of amino acids and their rates of synthesis from glucose-u-14c in rat brains during the preconvulsive state caused by administration of OMPm. 1 Following abbreviations were used: OMPm, 2-methyl-4-amino-5-hydroxymethylpyrimidine; GABA, ƒá-aminobutyric acid; TSC, thiosemicarbazide; PAL, pyridoxal. 245

246 FUKUSHIMA AND NOSE 1969 Under these experiments, we expected to know not only the changes of static levels of amino acids but also the changes of their rate of synthesis from glycolytic processes

2 246 FUKUSHIMA AND NOSE 1969 Under these experiments, we expected to know not only the changes of static levels of amino acids but also the changes of their rate of synthesis from glycolytic processes and to know whether there is or not the restoration of these changes with simultaneous administration of the vitamin. The results showed that the changes of the levels of brain amino acids were relatively small but the rates of incorporation of radioactivity into these amino acids were considerably inhibited by the administration of OMPm and no restora tive effect of the vitamin was observed on these changes, though the convulsion was completely protected. 1. Chemicals EXPERIMENTAL OMPm was kindly supplied by Dr. S. Yurugi, Research Laboratories, Takeda Chemical Company, Osaka. Thiosemicarbazide was the product of Wako Chemical Company. Amino acids and other chemicals were obtained from commercial sources. Glucose-U-14C was purchased from Daiichi Pure Chemicals Co., Ltd. A Nuclear Chicago Actigraph II was used to scan electrophoresis for radioactivity; a Nuclear Chicago Gas Flow Counter was also used for determinations of 14C radioactivities. 2. Preparation of Acid-Soluble Extracts of Rat Brain Wistar strain rats of both sexes, weighing g, were used. They were killed by immersion into liquid nitrogen and their brains were quickly chiselled out, homogenized in 10ml of ice-cold ethanol acidified with acetic acid. After centrifugation for 20min at 12,000rpm at 0, the sediment was resuspended in 3ml of acidified ethanol, recentrifuged, and the second supernatant was added to the first. The solution was evaporated in vacuo at 50 to dryness, dissolved in 10ml of distilled water, and centrifuged. The supernatant was washed twice with the same volume of ethyl ether and evaporated again to dryness. The residue was dissolved in water (at a ratio of 0.5ml/250mg dry weight of the brain) and analysed for individual amino acids after separation by high-voltage paper electrophoresis. 3. Isolation of Amino Acids by High-Voltage Paper Electrophoresis (17) Ten microliters of dissolved sample was applied with measured capillary on Whatman No, l paper (4 ~50cm) which had been soaked with pyridine-acetic acid water buffer, ph 3.6 (1:10:89). Paper electrophoresis was performed using the instrument of Sano-Type, Shiraimatsu, Osaka, with the same buffer mentioned above. The voltage loaded was about 1,000 volts/10cm of the paper. Two series of paper electrophoresis were performed, one was allowed to flow about 30min for separating GABA and aspartate, whereas the other about 200min for separating some other acidic or neutral amino acids and glucose. Identification of these amino acids were performed using thin-layer chromatography and co-chromatography by the same electrophoretic instrument. 4. Colorimetric Analysis for the Separated Amino Acids The dried paper was dipped in 0.5% ninhydrin in acetone and heated for 45 min at 65 for color development. The colored bands were cut out, eluted in 5ml of 70% ethanol, and the optical density was determined at 560mƒÊ. Standard color development was performed on the paper with 5, 10 and 20ƒÊl of 0.01M sodium

3 Vol. 15 EFFECT OF SEIZURE BY OMPM 247 glutamate at the same time in each color development. 5. Measurement of Radioactivity For the measurement of radioactivity of each amino acid, another electrophoresis was carried out, applying 0.1ml of the original sample divided on three papers (4 ~ 50cm) and the colored bands developed were eluted in 5ml of ethanol. An aliquot of the eluate was transferred to a planchet, dried and counted by Gas Flow Counter. The radioactivity of the original sample dissolved in 1ml of water in planchet was counted after drying. The radioactivity of the pellet was counted as follows. The dried pellet, after the dry weight being measured, was powdered and suspended in 70% ethanol (1ml/10mg of dried pellet) and a 1ml aliquot of the suspension was transferred to a planchet, dried and the radioactivity was counted. The absorp tion correction for true radioactivities was made by construction of an absorption curve for 14C as a function of thickness. 6. OMPm or Pyridoxine Treatment and Isotope Injection Rats were given an intraperitoneal or a subcutaneous injection of OMPm at a dose of 1mg/g of body weight. Another group of rats were given an intraperi toneal or a subcutaneous injection of TSC (15ƒÊg/g of body weight). TSC, the similar running fit-inducing substance, was used for comparison with the effect on amino acid levels in the brain. Glucose-U-14C, 1ml (10ƒÊCi) together with carrier glucose (1.3mg) was injected intraperitoneally 60min after administration of OMPm. All the rats including controls were sacrificed by immersion into liquid nitrogen 30min after isotope injection. For expecting restoration by vitamin B6, a part of each group of rats were given pyridoxal-hc1 simultaneously with OMPm or TSC at a dose of 20ƒÊg/g of body weight, a dose sufficient to prevent the seizure caused by OMPm or TSC. Color development with anisealdehyde sulfate reagent was used for the identification of glucose and the quantitative determination of glucose was performed by the method of Somogyi-Nelson (18). RESULTS 1. The Amino Acid Levels in Brain of Rats Treated with OMPm or TSC. The amino acid levels in rat brains at the beginning of running fit incited by OMPm or TSC injection were observed. The running fit of the rats treated with 1mg/g of OMPm occurred 126min after injection on an average and those treated with 15ƒÊg/g of TSC at 58min. Table 1 shows the levels of amino acids in rat brains at the beginning of running fit. The aspartate levels are not strictly showing the true levels because it may be contaminated with a part of glutathione which moved near aspartate electrophoretically. With the same reason, the levels of glutamine may contain threonine and a part of serine. However, the levels of aspartate and glutamine are not so far from the true values because of relatively high levels of these amino acids compared with others. The reduction of the level of glutamate and GABA was not manifest at the beginning (Table 1) and prior to the beginning of running fit (Table 2) induced by

4 248 FUKUSHIMA AND NOSE 1969 TABLE 1 Amino Acid Levels in Rat Brains at the Beginning of Running Fit Induced by OMPm or TSC The number of animals is given in parenthesis. 250 mg dry weight of the brain corresponds nearly to 1 g wet weight. Values are expressed as a mean }standard deviation. TABLE 2 Amino Acid Levels in Rat Brains Before the Initiation of Running Fit Treated with OMPm Values are expressed as a mean }standard deviation. The number of animals is given in parenthesis. OMPm. These results agree with other investigaters' data, using other convulsive agents (16, 19). In the case of TSC (Table 1), the reduction of glutamate was not observed and that of GABA was not so remarkable at the beginning of running fit compared with the case of OMPm though the convulsive action of TSC was somewhat stronger than that of OMPm. 2. Incorporation Studies of Glucose-U-14C into Brain Amino Acids of Rats The incorporation of 14C-glucose into individual amino acids were observed at the stage just before the beginning of running fit in order to exclude the secondary effect of convulsive movement. As the first running fit begins 120min after injection of 1mg/g of OMPm as described above, the incorporation study reveals the changes of preconvulsive stage. Fig. l shows a part of the actigram exhibiting the radioactivities of glutamate, glucose and glutamine. The band of glucose contains taurine. However, no labelling of taurine (19) was confirmed by thin-layer chromatography.

5 Vol. 15 EFFECT OF SEIZURE BY OMPM 249 FIG. 1 Actigrams of Labeled Amino Acids and Glucose Separated by High-Voltage Paper Electrophoresis The incorporation of radioactivity into glutamate and glutamane was considerably inhibited by the admin istration of OMPm as compared with the control. In contrast to the de creased incorporation, the radioacti vity of brain glucose was higher than the controls. It cannot be explained simply by the outcome of lowered incorporation of radioactive glucose into the brain itself, as shown in Table 3. The total incorporation of radio activity into the acid-soluble extract as well as the acid-insoluble preci pitate was considerably lowered by the administration of OMPm, and the decrease of incorporation in the soluble fraction was seen even by subtracting the radioactivity of 14C glucose remaining in the fraction. These results show the lowered utilization of 14C-glucose by the whole brain of the animals treated with OMPm. More important is the fact that these decreases led to the reduction of incorporation into other metabolites including the main four amino acids. In these observations, two or three possible changes in glucose utilization system of the brain caused by the agent might be considered; one is the effect on the circulatory system and the other on the membrane system involved in absorption or diffusion or both, of glucose at the site of peritoneal cavity, intima of blood vessels or blood-brain barrier. The changes in the enzyme systems concerning permeation, glycolytic process and of the citric acid cycle including decarboxylase TABLE 3 Incorpoation of Glucose-U-14C into Acid-Soluble Fraction and Percentage Distribution of Amino Acids Values are expressed as a mean of 5 or 6 animals. Radioactivies are counted for cpm/250mg dry weight of the brain.

250 FUKUSHIMA AND NOSE 1969 and transaminase may also be taken into consideration. It has been already reported that the absolute levels of brain glucose were not affected by OMPm administration (15).

6 250 FUKUSHIMA AND NOSE 1969 and transaminase may also be taken into consideration. It has been already reported that the absolute levels of brain glucose were not affected by OMPm administration (15). It was also found in this study. Therefore, the change in glucose radioactivity is expressed as the change in specific radioacti vity in Fig. 2. As for the incorporation of radioactivity into the individual amino acids, several remarkable findings were obtained; in the control group, markedly high incorporation of radioactivity into main amino acids including GABA, a functionally important amino acid, was found in total and specific radioactivities as shown in Fig. 2. At the same time, in the OMPm-treated group, a remarkable inhibition of the incorporation of 14C-glucose was observed in contrast with small changes of absolute levels of brain amino acids of rats treated with the same drug. These results might be significant for understanding the convulsive action of OMPm. In addition, unexpectedly, this inhibition could not be restored by simultaneous administration of PAL, though the dose of the vitamin completely prevented the seizure in the test animals. In Table 4, there are also some observable differences in the incorporation of 14C-glucose between the intraperitoneal and subcutaneous injections of OMP m. The cause of these differences is obscure. It may depend probably on the absorp tion rate of OMPm from the peritoneal cavity and from the subcutaneous tissues into the brain. When sacrificed, subcutaneous swelling at the injected site has not completely disappeared. The reduced absorption of glucose from the peritoneal surface which had been disturbed by previously medicated OMPm would be con sidered. It must be added finally that the specific radioactivity of GABA was unreasonably higher than that of glutamate. TABLE 4 Percentage Inhibiton of Specific Radioactivity DISCUSSION The slight reduction of the glutamate and GABA levels in the brain of the rat treated with OMPm was observed prior to the running fit, but these decreases are too small to ascribe to the causative change of the running fit leading to death of the animal. Moreover, these changes are not parallel to those observed in the case of TSC, a similar running fit-inducing drug. Meanwhile, fairly conspicuous change was observed in the actigram using 14C-glucose. If the metabolism of amino acids of the brain is directly correlated with convulsion, it is more important to consider the dynamic variabilites rather than the static levels of these amino acids, which are provably affected by convulsvie agents which compete with vitamin B6.

$increase in the radioactivity of glucose in the fraction. The radioactivities of main four amino acids are also decreased (30-70%) by OMPm injection.$

7 Vol. 15 EFFECT OF SEIZURE BY OMPM 251 The most remarkable change by OMPm is the decrease in the incorporation of 14C-glucose into metabolites in the soluble extract of brains in contrast to the increase in the radioactivity of glucose in the fraction. The radioactivities of main four amino acids are also decreased (30-70%) by OMPm injection. Though the absolute levels of glucose and of amino acids in each group were not so affected, the considerable differences in their radioactivities were found between normal and the OMPm-treated rat brains. In relation to this result, the difference of the rate of turnover of glucose metabolism might be considered. Generally, radioactive glucose in blood and all the tissues may gradually lower through dilution mechanism. In normal rats the labeled glucose injected into the peritoneal cavity shows its highest level in blood stream soon after injection. In all organs, the radioactivity of the glucose reach its highest level at first, followed by gradual lowering, and the radioactivity of each metabolite become continuously increased. The free glucose is converted into some other metabolites and re plenished from other glucose sourses which cause the fall of the radioactivity of glucose, but in the drug-treated rat, it is still in the state where the labeled glucose remains undiluted. It must be considered that there are some mechanisms which retard the rate of turnover in glucose metabolism by the administration of OMPm. This mecha nisms must be considered throughout the whole body. It may be explained by the decreased metabolism as a whole, or it may be metabolic inhibition at the particular site of glycolytic processes, but it could not be explained only by the difference in circulatory systems because of the reverse result of radioactivity between glucose and amino acids. It must be considered that there are some other mechanisms in glucose metabolism than circulatory changes by OMPm administration. It may be explained by the decreased metabolism as a whole, or it may be metabolic inhibition at the particular site of glycolytic processes. As for the amino acids, it is important to consider the dynamic change as a whole than the static levels of individual amino acids, because the decreased incor poration of 14C-glucose into amino acids as a whole is far more marked than the variances observed in the static levels of individual amino acids. In these cases, the percentage of glucose-radioactivity in total soluble extract is considerably high in the drug-treated rats in contrast with the control (Table 3). This percentage method of data of individual amino acids must be exhibited with the exception o radioactivity of glucose. The percentage radioactivity distributed into the individual amino acids were not so varied by the treatment of OMPm (Table 3). From this result, it is assumed that OMPm does not relate so much on the quantitative dis tribution of amino acids investigated; briefly, there is the parallel inhibition of the main amino acid synthesis (Table 4). There is also no decisive change in the absolute level of each amino acid investigated (Table 2). Concerning the GABA, a functionally important amino acid (25), a kind of compartmentalization (20, 21, 22) might be taken into consideration that the newly formed radioactive compartment of glutamate is metabolized quickly into GABA fraction which, as a whole, makes unreasonably higher specific radioactivity of GABA than that of glutamate, although it is another question concerned rigidly

8 252 FUKUSIIINIA AND NOSE 1969 with convulsive action of OMPm. The enzymatic inhibition at a particular site of amino acid metabolism that was found in vitro could not be considered always so important in vivo that the result obtained in vitro is not always showing the true state in living organisms, and that the conspicuously increased radioactive peak except glucose was not found throughout the course of actigram of separated soluble extract in drug-treated group. Though the difference in isotope incorporation between two groups of intraperitoneal and of subcutaneous injection of OMPm (Fig. 2, Tables 3, 4) may be the result of the difference of absorbed concentration of OMPm into the brain itself, as mentioned previously, and it may he related to the result of the in vitro experiments (10, 14), the running fit can be elicited in the same way after the same lucid interval so that this difference of isotopic incorporation may not be related to the true cause of the convulsion. That is the reason why the result of the experiment in vitro could not be related directly to the clinical symptoms that was elicited in viva administration of the agent. FIG. 2 R dioartivitirs of Isolated Amino Acids Observing the parcentage presentation of the incorporation (Table 3), considera ble part of the radioactivity of total soluble extract is due to the main four amino acids, so that, if it is limited within the brain itself, the metabolic changes occurring in amino acid synthesis cause great many effects on glucose metabolism. However, it must not be neglected that the decreased fraction other than the sum of main four amino acids (Table 3) is about (60%. Notwithstanding, there is no particular peak observed in the actigram except the main amino acids and glucose, so the specific site inhibiting each amino acid synthesis could not be important. The incorporation of glucose radioactivity into amino acids in liver is so little (23), that the amino acid synthesis reflects a part of glucose metabolism but the con siderable part of glucose metabolism is outside the amino acid metabolism. There fore, the delayed glucose metabolism cannot be explained only by the protracted synthesis of amino acids from the glycolytic process.m inard et a1, (19) suggested the diverted metabolism of pyruvate to some

9 Vol. 15 EFFECT OF SEIZURE BY OMPM 253 abnormal routes in the incorporation study of glucose. Speculatively in this study, it is partly because of decreased interconversion of free glucose to some other metabolites including the release of glucose from glucogen and from some other glucose sources, which makes the dilution of labeled glucose. There may be an involvement of phosphorylase activation (24) by OMPm in a way of abnormal catecholamine metabolism which is dependent on vitamin B6. At any rate, it must be important to separately consider the obtained metabolic changes induced by the drug and the true causal changes of convulsion. Moreover, the incorporation of radioactive glucose into individual amino acids is inhibited and the metabolism of free glucose itself is decreased by OMPm, but these changes could not be restored by the concomitant administration of PAL, though the running fit induced by OMPm could be overcome by PAL. The causative change of metabolism in running fit induced by OMPm was not elucidated, but it was found that there are great many metabolic changes caused by OMPm administration which conceal the true cause of OMPm convulsion. ACKNOWLEDGEMENT Great thanks to Prof. R. Iizuka of Department of Neuropsychiatry and Dr. A. Iwashima of Biochemical Institute for their kind advices and encouragements. REFERENCES 1. Makino, K., Kinoshita, T., Sasaki, T., and Shioi, T., Nature, 173, 34 (1954). 2. Makino, K., Kinoshita, T., Aramaki, Y., and Shintani, S., Nature, 174, 275 (1954). 3. Koike, M., Seikagaku, 26, 528 (1954); J. Vitaminol., 1, 254 (1955). 4. Makino, K., and Kinoshita, T., Vitamins, 8, 28 (1955). 5. Watanabe, A., and Shinya, S., Vitamins, 8, 517 (1955). 6. Torigoe, K., Viarnins, 9, 483 (1955). 7. Nishizawa, Y., and Kodama, T., and Kooka, T., J. Vitaminol., 3, 309 (1957). 8. Nishizawa, Y., Kodama, T., and Miyake, M., J. Vitaminol., 4, 1 (1958). 9. Nishizawa, Y., Kodama, T., and Hayashi, M., J. Vitaminol., 4, 132 (1958). 10. Makino, K., and Koike, M., Nature, 174, 1056 (1954). 11. Nishizawa, Y., Kodama, T., and Namba, S, J. Vitaminol., 4, 264 (1958). 12. Yamanaka, T., Vitamins, 13, 422 (1957). 13. Tomobe, W., Seikagaku, 30, 511 (1958). 14. Nishizawa, Y., J. Vitaminol., 5, 241 (1959). 15. Mandokoro, Y., Vitamins, 13, 161 (1957). 16. Rindi, G., and Ferrari, G., Nature, 183, 608 (1959). 17. Ohara, K., Advances in Neurological Sciences, 4, 523 (1960). 18. Somogyi, M., J. Biol. Chem., 195, 19 (1952). 19. Minard, F. N., and Mushahwar, I. K, J. Neurochein., 13, 1-11 (1966). 20. Ben, S., Lajtha, A., and Waelsch, H., J. Neurochem., 7, 186 (1961). 21. Takagaki, G., Ben, S., Clarke, D. D., Purpura, D. P., and Waelsch, H., Nature, 189, 326 (1961). 22. Cremer, J. E., J. Neurochem., 11, 165 (1964). 23. Gaitonde, M. K., Dahl, DR., and Elliott, K. A. C., Biocltem. J., 94, 345 (1965). 24. Klainer, L. M., Chi, Y. M., Freidberg, S. L., Rall, T. W., and Sutherland, E. W., J. Biol Chem., 237, 1239 (1962). 25. Mckhann, G M., Mickelsen, O., and Tower, D. B., Amer. J. Physiol., 200, 34 (1961).

OF TRANSAMINASE IN RAT TISUES

OF TRANSAMINASE IN RAT TISUES KOZO YAMADA, SHUNJI SAWAKI, AKIRA FUKUMURA AND MASARU HAYASHID epartment of Internal Mcdicine, Faculty of Medicine, Nagoya University, agoya Showa-ku, N (Received July 30,