0022-3042/79/ 100I-0977602.00/0
Jounml of Nuuroclwmrsrr) Vol. 33. pp. 977 to 979 Pergamon Preas Ltd 1979. Printed in Great Britain 0 International Society for Neurochemistry Ltd
SHORT COMMUNICATION
Regional distribution of folate in human brain (Received 17 April 1979. Accepted 27 April 1979)
LITTLEis known about the role of folate in nervous tissue, although it may be involved in nucleic acid synthesis (HALTIA, 1970), methylation of monoamines (LADURON.1972) or synaptic events (BRIDGERS & MCCLAIN,1972). Moreover, there has been no report on regional distribution of folate in human brain. We determined the concentration of folate in 22 parts of human brain obtained at autopsy, since the knowledge of regional distribution is necessary as a first step to an insight into some important roles of folate in brain function. This study disclosed that there were remarkable differences in the folate level among various structures in human brain.
5&150mg, were weighed and homogenized in 1"; Na ascorbate, pH 7.0 (30 mg tissue per ml). The homogenate was then heated in a boiling water bath for 7min. After centrifuging at 3500 rev./min (2000 g) for 20 min, the supernatant was transferred to a graduated test tube to measure the volume. Subsequently, it was stored at -20°C for less than 2 days until analysed. A '''I folate radioassay kit purchased from Clinical Assays. Massachusetts, U.S.A. was used for the determination of folate. The assay was carried out according to the manufacturer's directions, using 0.1 ml aliquots of the supernatants.
RESULTS
MATERIALS AND METHODS As a preliminary study, post-mortem changes in the concentration of folate were estimated in rat brain over a period of 9 h. Male Wistar rats weighing about 200 g were divided into four groups of three rats each. The first three rats were killed by dropping into liquid nitrogen. The brain was removed from the skull in a frozen state and stored at -80°C. Three other rats were decapitated, and the heads left at room temperature for 3, 6 and 9 h, respectively, when the brains were removed and transferred to -80°C. The assay of folate was performed as described below. No statistically significant differences were found in the folate content in brain among the four groups of rats. Therefore, it was assumed that in the case of human brain as well there would be no substantial changes in the folate content until at least 9 h after death. Brains were obtained at autopsy from a 55-year-old man (presumed age), a 67-year-old woman and a 61-year-old man. These had died of acute alcoholism. carcinoma of the rectum and carcinoma of the pancreas, respectively, without evidence of neurological symptoms. The exact age and history of the first case were not known. The intervals between death and autopsy were about 10, 9 and 2.5 h, respectively. At autopsy, the brain was cut in two sagittally, and the right cerebral and cerebellar hemispheres with the right half of the brain stem were stored at -80°C. Prior to the dissection of various regions, the frozen half-brain was allowed to stand at room temperature until it became less hard, and then dissected into 22 parts in a semifrozen state, occasionally placing them close to blocks of dry ice to prevent them from becoming too soft. No gross abnormalities were found in any part of the brain. The extraction of folate from tissue was performed according to the method by SHINet al. (1974) which was slightly modified. The dissected portions of brain, K e y words: folate; human brain.
As shown in the Table 1, distinct differences were
detected in the content of folate between regions of human brain. Although the absolute amount was often variable, there was a fairly constant order of concentration common to the three cases examined. In grey matter, the highest value in the average was observed in the mammillary body, followed by the amygdala. caudate nucleus, dentate nucleus, superior temporal gyrus and cerebellar cortex, all of which contained more than 400ng folate per g wet wt of tissue. In white matter, the greatest concentrations of folate were present in the optic tract, corpus callosum, crus cerebri and superior temporal gyrus, in descending order of concentration: these areas contained more than 200 ng folate per g wet wt. In the four areas of the cerebral cortex, the temporal cortex almost always showed the highest value as compared with the frontal, parietal and occipital cortices. It should also be stressed that there were considerable differences in the folate level of white matter among the regions examined. In general, the folate content was higher in grey matter than in white matter, as seen in the cerebral cortex and its adjacent white matter. But some white matter showed comparatively high levels of folate, as observed in the corpus callosum and crus cerebri.
DISCUSSION The present study revealed a unique distribution of folate in human brain in which the mammillary body had the highest concentration among 22 regions examined. The folate level of the amygdala and optic tract were also high. KOREVAAR et al. (1973) assayed the level of 5-methyltetrahydrofolate in rat brain and found the highest value to be in the midbrain raphe nuclei and next in the corpus striatum with relatively low values in the hypothalamus and hypocampus, and almost no measurable amount in the substantia nigra. These are considerably different from our results. However, a strict comparison would not be
977
Short communication
978
TABLE 1. FOLATECONCENTRATION
Precentral gyms G Precentral gyrus W Postcentral gyrus G Postcentral gyms W Superior temporal gyrus G Superior temporal gyrus W Banks of calcarine sulcus G Banks of calcarine sulcus W Corpus callosum Thalamus Mammillary body Putamen Globus pallidus Caudate nucleus Internal capsule Amygdala Substantia nigra Red nucleus Crus cerebri Cerebellar cortex Dentate nucleus Optic tract
I N REGIONS OF HUMAN BRAIN
Case 1
Case 2
Case 3
203 63 217
49 1 189 397 260 452 184 259 147 561 282 769 310 413 638 192 640 436 459 376 538 581 811
324 96 239 86 474 300 310 101 242 38 1 693 175 21 1 397 127 784 24 1 350 218 352 224 47 1
-
343 -
25 1 -
306 339 824 196 303 449 72 ~
279 23 1 265 347 538 766
Mean f S.D. 339 118 116 2 53 284 & 80 173 k 87 423 & 51 242 & 58 273 t 26 124 & 23 370 f 138 334 f 41 762 54 227 k 59 309 83 495 k 104 130 49 712 k 72 319 t 84 347 k 93 286 5 66 412 k 89 448 k 159 683 k 151
+
Values are expressed as ng per g wet wt of tissue. G, grey matter; W, white matter. The age, sex, cause of death and elapsed time between death and autopsy of cases 1. 2 and 3 were: 55-year-old man (presumed age), acute alcoholism, 10 h; 67-year-old woman, carcinoma of the rectum, 9 h, and 61-year-old man, carcinoma of the pancreas, 2.5 h, respectively. A competitive binding radioassay was performed for the determination of folate. proper, since we measured the total amount of folate de- pecially on the difference between motor and sensory nerve rivatives, including 5-methyltetrahydrofolate. fibres. This will be the subject of our next study. The distribution of 5-methyltetrahydrofolate in brain might have some correlation with serotonergic nerve ter- Acknowledgements-We are grateful to Prof. K. AKAI and minals, as was suggested by KOREVAAR et al. (1973). HowDr. Y. KAWAGUCHI, Department of Pathology, Kyorin ever, the total folate has a different distribution which University School of Medicine for their co-operation. seems not to correspond to the distribution of any of the monoamines (FUXE, 1965; GLOWINSKI & BALDESSARINI,The First Department of Y . YOSHINO 1965; HORNYKIEWICZ, 1966). This may suggest diverse H. KOIKE Internal Medicine, roles of folate derivatives in brain tissue. Folate might par- Kyorin University School of Medicine, Y.WAKABAYASHI ticipate in nucleic acid synthesis (HALTIA,1970), and the Mitaka, Tokyo. Japan Y. SAWAGUCHI relevance of the latter to the process of learning was reported by HYDEN& EGYHAZI(1962). Therefore, the high level of folate in the mammillary body might have some bearing on that aspect of cerebral function (CAMPER, 1928), REFERENCES although a recent study on the memory (HOREL,1978) sugW. F. & MCCLAINL. D. (1972) Some interrelagested that temporal cortex and its connections were the BRIDGERS tionships of pyridoxal phosphate, folk acid, and serine most important, without assigning an essential role to the metabolism in brain, in Advances in Biochemical Psychomammillary body. In our study the temporal cortex P., eds.) Vol. 4, pharmacology (COSTAE. & GREENCARD showed a comparatively high level of folate among the pp. 81-92. Raven Press, New York. four regions of the cerebral cortex. Also to be emphasized in our results are the differences FUXEK. (1965) The distribution of monoamine terminals in the central nervous system. Actu physiol. scand. 64, in the folate level of white matter in the several regions examined. The white matter dissected from the four regions SUPPI.247, 37-85. of the cerebral cortex might have contained a small GAMPER E. (1928) Zur Frage der Polioencephalitis haeamount of adjacent grey matter, causing some contamimorrhagica der chronischen Alkohliker. Anatomische nation. But this artifact is unlikely in the case of the optic Befunde beim alkoholischen Korsakow und ihre Beziehungen zum klinischen Bild. Dt. 2. NeruHeilk. 102, tract, corpus callosum, internal capsule and crus cerebri which consist entirely of white matter. The reason for the 122-129. uneven distribution of folate in various regions of white GLOWINSKI J. & BALDESSARINI R. J. (1965) Metabolism of norepinephrine in the central nervous system. Pharmac. matter is unknown, but it may be suggested that the folate content of nerve axons is different, depending on the funcRev. 18, 1201-1238. tion of the nerve fibre. The finding that the optic tract HALTIA M. (1970) The effects of folate deficiency on neuronal RNA content. A quantitative cytochemical study. contains a high amount of folate also indicates the necesBr. J . exp. Path. 51, 191-196. sity of further study on other peripheral nerve fibres. es-
Short communication
HORELJ . A. (1978) The neuroanatomy of amnesia. A critique of the hippocampal memory hypothesis. Brain 101, 403445. HORNYKIEWICZ 0. (1966) Dopamine (3-hydroxytryptamine) and brain function. Pharmac. Rev. 18, 925-964. HYD~N H. & EGYHAZIE. (1962) Nuclear RNA changes during a learning experiment in rats. Proc. natn. Acad. Sci., U.S.A. 48, 1366-1373. KOREVAAR W. C., GEYER' M. A,, KNAPP S., Hsu L. L. & MANDELL A. J. (1973) Regional distribution of 5-methyl-
979
tetrahydrofolic acid in brain. Nature, New Bid. 245, 244245. LADURONP. (1972) N-Methylation of dopamine to epinine in brain tissue using N-methyltetrahydrofolic acid as the methyl donor. Nature, New Biol. 238, 212-213. SHINY. S., BUEHRINC K. U. & STOKSTAD E. L. R. (1974) Studies of folate compounds in nature. Folate compounds in rat kidney and red blood cells. Archs Biochern. Biophys. 163, 21 1-224.
Jounml of Nuuroclwmrsrr) Vol. 33. pp. 977 to 979 Pergamon Preas Ltd 1979. Printed in Great Britain 0 International Society for Neurochemistry Ltd
SHORT COMMUNICATION
Regional distribution of folate in human brain (Received 17 April 1979. Accepted 27 April 1979)
LITTLEis known about the role of folate in nervous tissue, although it may be involved in nucleic acid synthesis (HALTIA, 1970), methylation of monoamines (LADURON.1972) or synaptic events (BRIDGERS & MCCLAIN,1972). Moreover, there has been no report on regional distribution of folate in human brain. We determined the concentration of folate in 22 parts of human brain obtained at autopsy, since the knowledge of regional distribution is necessary as a first step to an insight into some important roles of folate in brain function. This study disclosed that there were remarkable differences in the folate level among various structures in human brain.
5&150mg, were weighed and homogenized in 1"; Na ascorbate, pH 7.0 (30 mg tissue per ml). The homogenate was then heated in a boiling water bath for 7min. After centrifuging at 3500 rev./min (2000 g) for 20 min, the supernatant was transferred to a graduated test tube to measure the volume. Subsequently, it was stored at -20°C for less than 2 days until analysed. A '''I folate radioassay kit purchased from Clinical Assays. Massachusetts, U.S.A. was used for the determination of folate. The assay was carried out according to the manufacturer's directions, using 0.1 ml aliquots of the supernatants.
RESULTS
MATERIALS AND METHODS As a preliminary study, post-mortem changes in the concentration of folate were estimated in rat brain over a period of 9 h. Male Wistar rats weighing about 200 g were divided into four groups of three rats each. The first three rats were killed by dropping into liquid nitrogen. The brain was removed from the skull in a frozen state and stored at -80°C. Three other rats were decapitated, and the heads left at room temperature for 3, 6 and 9 h, respectively, when the brains were removed and transferred to -80°C. The assay of folate was performed as described below. No statistically significant differences were found in the folate content in brain among the four groups of rats. Therefore, it was assumed that in the case of human brain as well there would be no substantial changes in the folate content until at least 9 h after death. Brains were obtained at autopsy from a 55-year-old man (presumed age), a 67-year-old woman and a 61-year-old man. These had died of acute alcoholism. carcinoma of the rectum and carcinoma of the pancreas, respectively, without evidence of neurological symptoms. The exact age and history of the first case were not known. The intervals between death and autopsy were about 10, 9 and 2.5 h, respectively. At autopsy, the brain was cut in two sagittally, and the right cerebral and cerebellar hemispheres with the right half of the brain stem were stored at -80°C. Prior to the dissection of various regions, the frozen half-brain was allowed to stand at room temperature until it became less hard, and then dissected into 22 parts in a semifrozen state, occasionally placing them close to blocks of dry ice to prevent them from becoming too soft. No gross abnormalities were found in any part of the brain. The extraction of folate from tissue was performed according to the method by SHINet al. (1974) which was slightly modified. The dissected portions of brain, K e y words: folate; human brain.
As shown in the Table 1, distinct differences were
detected in the content of folate between regions of human brain. Although the absolute amount was often variable, there was a fairly constant order of concentration common to the three cases examined. In grey matter, the highest value in the average was observed in the mammillary body, followed by the amygdala. caudate nucleus, dentate nucleus, superior temporal gyrus and cerebellar cortex, all of which contained more than 400ng folate per g wet wt of tissue. In white matter, the greatest concentrations of folate were present in the optic tract, corpus callosum, crus cerebri and superior temporal gyrus, in descending order of concentration: these areas contained more than 200 ng folate per g wet wt. In the four areas of the cerebral cortex, the temporal cortex almost always showed the highest value as compared with the frontal, parietal and occipital cortices. It should also be stressed that there were considerable differences in the folate level of white matter among the regions examined. In general, the folate content was higher in grey matter than in white matter, as seen in the cerebral cortex and its adjacent white matter. But some white matter showed comparatively high levels of folate, as observed in the corpus callosum and crus cerebri.
DISCUSSION The present study revealed a unique distribution of folate in human brain in which the mammillary body had the highest concentration among 22 regions examined. The folate level of the amygdala and optic tract were also high. KOREVAAR et al. (1973) assayed the level of 5-methyltetrahydrofolate in rat brain and found the highest value to be in the midbrain raphe nuclei and next in the corpus striatum with relatively low values in the hypothalamus and hypocampus, and almost no measurable amount in the substantia nigra. These are considerably different from our results. However, a strict comparison would not be
977
Short communication
978
TABLE 1. FOLATECONCENTRATION
Precentral gyms G Precentral gyrus W Postcentral gyrus G Postcentral gyms W Superior temporal gyrus G Superior temporal gyrus W Banks of calcarine sulcus G Banks of calcarine sulcus W Corpus callosum Thalamus Mammillary body Putamen Globus pallidus Caudate nucleus Internal capsule Amygdala Substantia nigra Red nucleus Crus cerebri Cerebellar cortex Dentate nucleus Optic tract
I N REGIONS OF HUMAN BRAIN
Case 1
Case 2
Case 3
203 63 217
49 1 189 397 260 452 184 259 147 561 282 769 310 413 638 192 640 436 459 376 538 581 811
324 96 239 86 474 300 310 101 242 38 1 693 175 21 1 397 127 784 24 1 350 218 352 224 47 1
-
343 -
25 1 -
306 339 824 196 303 449 72 ~
279 23 1 265 347 538 766
Mean f S.D. 339 118 116 2 53 284 & 80 173 k 87 423 & 51 242 & 58 273 t 26 124 & 23 370 f 138 334 f 41 762 54 227 k 59 309 83 495 k 104 130 49 712 k 72 319 t 84 347 k 93 286 5 66 412 k 89 448 k 159 683 k 151
+
Values are expressed as ng per g wet wt of tissue. G, grey matter; W, white matter. The age, sex, cause of death and elapsed time between death and autopsy of cases 1. 2 and 3 were: 55-year-old man (presumed age), acute alcoholism, 10 h; 67-year-old woman, carcinoma of the rectum, 9 h, and 61-year-old man, carcinoma of the pancreas, 2.5 h, respectively. A competitive binding radioassay was performed for the determination of folate. proper, since we measured the total amount of folate de- pecially on the difference between motor and sensory nerve rivatives, including 5-methyltetrahydrofolate. fibres. This will be the subject of our next study. The distribution of 5-methyltetrahydrofolate in brain might have some correlation with serotonergic nerve ter- Acknowledgements-We are grateful to Prof. K. AKAI and minals, as was suggested by KOREVAAR et al. (1973). HowDr. Y. KAWAGUCHI, Department of Pathology, Kyorin ever, the total folate has a different distribution which University School of Medicine for their co-operation. seems not to correspond to the distribution of any of the monoamines (FUXE, 1965; GLOWINSKI & BALDESSARINI,The First Department of Y . YOSHINO 1965; HORNYKIEWICZ, 1966). This may suggest diverse H. KOIKE Internal Medicine, roles of folate derivatives in brain tissue. Folate might par- Kyorin University School of Medicine, Y.WAKABAYASHI ticipate in nucleic acid synthesis (HALTIA,1970), and the Mitaka, Tokyo. Japan Y. SAWAGUCHI relevance of the latter to the process of learning was reported by HYDEN& EGYHAZI(1962). Therefore, the high level of folate in the mammillary body might have some bearing on that aspect of cerebral function (CAMPER, 1928), REFERENCES although a recent study on the memory (HOREL,1978) sugW. F. & MCCLAINL. D. (1972) Some interrelagested that temporal cortex and its connections were the BRIDGERS tionships of pyridoxal phosphate, folk acid, and serine most important, without assigning an essential role to the metabolism in brain, in Advances in Biochemical Psychomammillary body. In our study the temporal cortex P., eds.) Vol. 4, pharmacology (COSTAE. & GREENCARD showed a comparatively high level of folate among the pp. 81-92. Raven Press, New York. four regions of the cerebral cortex. Also to be emphasized in our results are the differences FUXEK. (1965) The distribution of monoamine terminals in the central nervous system. Actu physiol. scand. 64, in the folate level of white matter in the several regions examined. The white matter dissected from the four regions SUPPI.247, 37-85. of the cerebral cortex might have contained a small GAMPER E. (1928) Zur Frage der Polioencephalitis haeamount of adjacent grey matter, causing some contamimorrhagica der chronischen Alkohliker. Anatomische nation. But this artifact is unlikely in the case of the optic Befunde beim alkoholischen Korsakow und ihre Beziehungen zum klinischen Bild. Dt. 2. NeruHeilk. 102, tract, corpus callosum, internal capsule and crus cerebri which consist entirely of white matter. The reason for the 122-129. uneven distribution of folate in various regions of white GLOWINSKI J. & BALDESSARINI R. J. (1965) Metabolism of norepinephrine in the central nervous system. Pharmac. matter is unknown, but it may be suggested that the folate content of nerve axons is different, depending on the funcRev. 18, 1201-1238. tion of the nerve fibre. The finding that the optic tract HALTIA M. (1970) The effects of folate deficiency on neuronal RNA content. A quantitative cytochemical study. contains a high amount of folate also indicates the necesBr. J . exp. Path. 51, 191-196. sity of further study on other peripheral nerve fibres. es-
Short communication
HORELJ . A. (1978) The neuroanatomy of amnesia. A critique of the hippocampal memory hypothesis. Brain 101, 403445. HORNYKIEWICZ 0. (1966) Dopamine (3-hydroxytryptamine) and brain function. Pharmac. Rev. 18, 925-964. HYD~N H. & EGYHAZIE. (1962) Nuclear RNA changes during a learning experiment in rats. Proc. natn. Acad. Sci., U.S.A. 48, 1366-1373. KOREVAAR W. C., GEYER' M. A,, KNAPP S., Hsu L. L. & MANDELL A. J. (1973) Regional distribution of 5-methyl-
979
tetrahydrofolic acid in brain. Nature, New Bid. 245, 244245. LADURONP. (1972) N-Methylation of dopamine to epinine in brain tissue using N-methyltetrahydrofolic acid as the methyl donor. Nature, New Biol. 238, 212-213. SHINY. S., BUEHRINC K. U. & STOKSTAD E. L. R. (1974) Studies of folate compounds in nature. Folate compounds in rat kidney and red blood cells. Archs Biochern. Biophys. 163, 21 1-224.
