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Biogenic amine levels in the mid-term human fetus
Developmental Brain Research, 7 (1983) 9-12 Elsevier Biomedical Press
9
Biogenic Amine Levels in the Mid-Term Human Fetus NAYYAR A. MASUDI* and DESMOND P. GILMORE** Institute of Physiology, The University, Glasgow G12 8QQ, Scotland ( U.K.) (Accepted June 29th, 1982) Key words: fetal brain - - CSF - - biogenic amine concentrations
Spectrophotofluorometric assays were used to measure concentrations of DA, NA, 5-HT and 5-HIAA present in extracts of brain tissue and CSF taken from midterm gestational fetuses of both sexes. The presence of biogenic amines was demonstrated in fetuses as early as 10.5 weeks gestation. In the hypothalamus, the concentrations (expressed in ng/100 mg tissue) ranged from 10.2 to 62.2 for NA, 11.6 to 258.8 for DA, 7.6 to 38.6 for 5-HT and 56.6 to 114.1 for 5-HIAA. Concentrations of DA and 5-HT were found to be significantly(P < 0.05) higher in the hypothalamus than in the cortex, but those of NA and 5-HIAA were similar in both areas. No sex differencewas observed. The concentrations of biogenicamines in the CSF (expressed in ng/ml) were 63.1-286.7 for NA, 43.2-108.1 for DA, 30.9-87.5 for 5-HT and 8.0-42.8 for 5-HIAA. INTRODUCTION It has recently been shown 15 that the catecholamines and serotonin (5-HT) play a role in the androgen-dependent sexual differentiation of the hypothalamus in rats and it is possible that in the human too, they serve a similar function. However, little knowledge at present exists about the concentrations of the different biogenic amines in the brain during human fetal development. In the present study concentrations of noradrenaline (NA), dopamine (DA), 5-HT and 5-hydroxyindole acetic acid (5-HIAA) were measured in the brain and cerebrospinal fluid (CSF) of mid-term human fetuses. MATERIALS AND METHODS Human fetuses were collected immediately after their removal from the uterus by hysterotomy (performed for gynaecosocial reasons under general anaesthesia). They were packed in ice and brought to the Institute of Physiology, where dissection was carried out. The time on average between hystero-
tomy termination and freezing of the tissue in liquid nitrogen was less than 45 min. Fetal sex was initially determined by examination of the external genitalia and subsequently confirmed by histological examination o f the gonads. Cord serum was collected whenever possible, and CSF was aspirated by lumbar puncture. These were frozen in liquid nitrogen and stored at - - 2 0 °C. Crown-rump length was measured and the fetuses were weighed. These measurements were compared with those in the Table published by Iffy et alA ° for age assessment. The brain was exposed by first making an incision across the frontal bones of the skull. The hypothalamus and a piece of cortical tissue from the frontal lobe were removed and separately frozen in liquid nitrogen. These samples were also stored at --20 °C until assayed. The material examined in this study included the hypothalamus and a piece of frontal lobe from 33 fetal brains, 5 whole brains (from smaller fetuses where removal of the hypothalamus was not possible), and CSF samples from 16 fetuses of midterm gestational age. Cord serum was obtained from
* Present address: Physiology Department, Sind Medical College, Jinnah Postgraduate Medical Centre, J.P.M.C. Campus, Karachi-35, Pakistan. ** To whom correspondence and reprint requests should be addressed. 0165-3806/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
10 a total of 32 male and 21 female fetuses and the testosterone and oestradiol in it measured by radioimmunoassay according to the procedure outlined by Cook et al. 6. Weighed pieces of brain were • ' / allowed to thaw and then homogentzed m 0.5 O,% HC1 following a similar procedure to that described by Campbell et al. 4. The final volume of the extracts was adjusted so that 1.0 ml of extract was equivalent to 160 mg of brain tissue. In these 1.0 ml brain tissue extracts, and in 1.0 ml CSF samples, NA and DA concentrations were measured using slight modifications of the method described by Laverty and Taylor 11. The procedure followed by Snyder et al. t8 was used to estimate concentrations of 5-HT in the samples and that of Udenfrend et al. 19 for 5-HIAA. A small number of extracts and CSF samples were purified by being chromatographed on a Bio-Gel P~ column (90 × 1.5 cm). The gel used had a 200-400 mesh size. The eluant buffer was Na H~PO4 (0.05 M, pH 7.5). H u m a n serum albumin was added to the eluarU to prevent non-specific binding to the gel or to the glass of the column. The flow rate was 6 ml/h and the column was run at a temperature of 4 °C. A total of 200 1.0 ml fractions were collected from each column run. RESULTS The results of the non-eluted extracts and CSF samples are summarized in Table I and those eluted on the Bio-Gel P2 column in Table II. Values (corrected for recovery) recorded for the different amines are expressed in ng/100 mg wet weight of brain tissue and ng/ml of CSF. Differences were analyzed statistically by Student's t-test. F r o m the non-eluted extracts and CSF samples
recoveries of internal standards were 70'~,i for NA, 68'~o for DA, 65 !V,,for 5-HT and 75'%~I for 5-HIAA. It can be seen that although there are significantly higher levels (P < 0.05) of DA and 5-HT in the hypothalamus compared to the cortex, the hypothalamic and cortical concentrations of" NA were not significantly different. Concentrations of 5H I A A in the cortex and hypothalamus were also similar. Concentrations of NA. DA, 5-HT and 5H I A A in the CSF of mid-term fetuses showed no significant sex differences. The concentration of 5HIAA in the human fetal CSF found in this study (19. I :k 3.2 ng/ml) was in agreemenl with a previous report 20 which showed that 5-HIAA concentrations in the adult human lumbar CSF were 22.0 ! t2.4 ng/ml and in the human cisternal CSF 27.7 i- 13.0 ng/ml. Concentrations of biogenic amines covering the area of each peak identified by its native fluorescence in the eluted fractions of human fetal brain tissue extractions and CSF samples from the BioGel Pz columns are presented in Table 1I. Recoveries of internal standards for NA, DA and 5-HT in the eluted fractions of hypothalamic extracts were () ( ) ,' 81.3y,',, 63.3/~, and 63.00/o respectively. Internal standard recoveries in the eluted fractions of whole brain extracts were 83 ~ for NA, 95 "~ for D A and 60 °j for 5-HT, while recoveries of" the internal /o standards in the CSF samples were 60°'~ for NA, 81 o~ for DA and 73 ~,/, for 5-HT. No significant differences in NA, DA and 5-HT concentrations (Table lI) were observed between the whole brain tissue hypothalamic and CSF samples run through the Bio-Get P2 columns, although their presence was again quantitatively demonstrated.
TABLE 1 Concentrations of NA, DA, 5-HT and 5-HIAA in the mid-term fetus ( ~ ± S.E.M.)
Cortex(ng/10Omg) Range Hypothalamus(ng/10Omg) Range CSF (ng/ml) Range
NA
DA
5-HT
5-HIAA
35.1 ~+_ 5.1 (19) (6.8-79.2) 34.4 ± 3.0 (21) (10.2-62.2) 143.8 3- 20.9 (11) (63 •1-286.7)
73.t 3_ 7.6 (17) (18.0-123.9) 118.1 ~ 16.9 (21)* (11.6-258.8) 70.4 L 5.6 (11) (43.2-108.1)
14.0 -t: 1.8 (17) (3.2-31.2) 21.0 ~ 3.1 (12)* (7.6-38.6) 60.2 :~ 4.8 (1l) (30.9-87.5)
75.9 :!- 4.0 (12) (48.5--95.5) 79.0 4.8 (12) (56.6-114.1 ) 19.1 3.2(11) (8.0:-42.8)
* Significantly higher than in cortex, P • 0.05.
11 TABLE II Concentrations of biogenic amines in the eluted fractions of human fetal brain tissue extracts run through Bin-Gel P~ columns ( ~ ± S.E.M.)
Whole brain (ng/100 mg) Hypothalamus (ng/100 mg) CSF (ng/ml)
?CA
DA
5-HT
151.0 ± 6.8 (5) 126.2 ± 12.8 (5) 175.4 ± 7.3 (4)
141.3 ± 14.8 (5) 131.4 ± 15.9 (5) 58.1 ± 7.4 (4)
33.5 ± 3.6 (5) 31.0 ± 3.1 (5) 38.7 ± 4.6 (4)
The radioimmunoassay for testosterone showed an intraassay variation of 9 ~ and an interassay variation of 12 ~. The variations for oestradiol were 14 ~ and 17 ~ respectively. Levels of oestradiol and testosterone in the cord serum were very similar to those recorded by other workers 14. These were 4.9 :k 0.6 ng/ml (n --~ 32) in the male and 1.2 4- 0.2 (n ---- 21) in the female. The higher serum testosterone values, up to 16.7 ng/ml, were seen in fetuses between 11 and 18 weeks of age; after this time testosterone levels declined reflecting the falling levels of hCG and thus the reduced stimulus to the interstitial Leydig cells. Human fetal oestradiol concentrations showed no significant sex difference, being 1.7 ± 0.3 ng/ml (n = 13) in the male and 1.8 4- 0.4 ng/ml (n ~ 10) in the female. DISCUSSION The spectrophotofluorometric methods used in this study were able to demonstrate clearly the presence of biogenic amines in the mid-term fetus and also measure their concentrations in the different brain tissues and CSF. The methods used were found to be both sensitive and accurate and could detect biogenic amine concentrations of as low as 1-10 ng/100 mg tissue. Observations made in this study upon the hypothalamic concentrations of catecholamines and indoleamines are from the whole hypothalamus and not from individual nuclei. In contrast, much recent work has been carried out on the rat and centered upon the concentrations of DA, NA, 5-HT and 5-HIAA in specific regions of the hypothalamus and braina,S,la,16,aT. In primates the concentrations of DA, N A , 5-HT and 5-HIAA in the hypothalamus and other parts of the rhesus monkey brain have been also reported 2. More recently the levels of these substances were also in-
vest±gated in the adult human brain 5. In the present study the presence of biogenic amines was detected and measured in fetuses of as early as 10.5 weeks gestational age confirming the observations of a previous finding 9. The concentration of NA in the whole human fetal brain (10.5-13.5 weeks gestational age) was found to be 209 ng/100 mg of tissue while that of DA was 106 ng/100 mg. The ratio of NA/DA was 1.98. This agrees closely with NA/DA ratio of 2 measured in the brain of a single 20-week-old fetus 1. The whole brain catecholamine levels in 3 fetuses of 12-16 weeks gestational age were determined and found to be very low 12. A mean of 244 ng of NA and 101 ng of DA was found per brain, corresponding to a concentration of only 18 ng and 9 ng/g brain tissue respectively12. However, the NA/DA ratio was 2 which is again in good agreement with the pattern obtained in this study. In the hypothalamic extracts the NA and DA concentrations were found, as might be expected, to be different from those in the brain extracts. In the human fetal hypothalamus NA levels were 34 ng/100 mg of tissue while DA levels were 118 ng/100 mg of tissue. The NA/DA ratio here was 0.3. This is not dissimilar to the ratio of 0.46 for the rat fetal whole brain of 17 days gestation 7. One striking feature resulting from the present investigation was that 5-HT concentrations in the human fetal brain tissue were found to be higher than those previously described for the adult. The concentrations of 5-HT in the adult human hypothalamus was 10 ng/100 mg and in the cortex 1.05 ng/100 mg tissue 5. This contrasts with the values of 21 ng/100 mg in the fetal hypothalamus and 35 ng/100 mg in the fetal cortex recorded in the present study. The concentrations of 5-HIAA found in the hypothalamus (79 ng/100 rag) and cortex (73 ng/100 rag)
12 of h u m a n fetuses were very similar a n d n o t significantly different f r o m the values reported for the
ACKNOWLEDGEMENTS
adult rat h y p o t h a l a m u s a n d b r a i n 8,17. The concen-
We t h a n k Professor M. C. M a c N a u g h t o n , Dr.
trations of 5 - H I A A in the h u m a n fetal C S F samples
A n d r e w Calder a n d their staff at Glasgow Royal
(19 ng/ml) recorded in the present study, were also
Infirmary for providing hysterotomy specimens; Dr. Brian Cook for carrying out testosterone a n d
similar to those reported for adult h u m a n l u m b a r C S F values (22 ng/ml). The results of the present study for 5 - H I A A c o n c e n t r a t i o n s are also in good agreement with the findings of Brown et al. e, who showed a similar p a t t e r n of 5 - H I A A c o n c e n t r a t i o n s in the m o n k e y brain. Moreover, the presence of 5H I A A in the h u m a n fetuses clearly indicates the rapid b r e a k d o w n of 5-HT in the b r a i n tissue.
REFERENCES 1 Bertler, A., Occurrence and localization of catecholamines in the human brain, Acta physiol, seand., 51 (1961) 97-107. 2 Brown, R. M., Crane, A. M. and Goldman, P. S., Regional distribution of monoamines in the cerebral cortex and subcortical structures of the rhesus monkey: concentrations and in vivo synthesis rates, Brain Res., 168 (1979) 133-150. 3 Brownstein, M. J., Palkovits, M., Saavedra, J. M. and Kizer, J. S., Distribution of hypothalamic hormones and neurotransmitters within the diencephalon. In L. Martini and W. F. Ganong (Eds.), Frontiers in Neuroendocrinology, Vol. 4, Raven Press, New York, 1976, pp. 1-23. 4 Campbell, H. J., Feuer, G. and Harris, G. W., The effect of intra-pituitary infusion of median eminence and other brain extracts on anterior pituitary gonadotropic secretion, J. Physiol. (Lond.), 170 (1964) 474-486. 5 Carlsson, A., Adolfsson, R., Aquilonius, S., Gottfries, C., Oreland, L., Svennerholm, L. and Winblad, B., Biogenic amines in human brain in normal aging, senile dementia and chronic alcoholism. In Goldstein et al. (Eds.), Ergot Compounds and Brain Function: Neuroendocrine and Neuropsychiatric Aspects, Raven Press, New York, 1980, pp. 295-304. 6 Cook, B., Hunter, R. H. F. and Kelly, A. S. L., Steroidbinding proteins in follicular fluid and peripheral plasma from pigs, cows and sheep, J. Reprod. Fert., 51 (1977) 65-71. 7 Coyle, J. T., and Henry, D., Catecholamines in fetal and newborn rat brain, J. Neurochem., 21 (1973) 61~7. 8 H6ry, M., Laplante, E. and Kordon, C., Participation of serotonin in the phasic release of luteinizing hormone. 1i. Effects of lesions of serotonin-containingpathways in the central nervous system, Endocrinology, 102 (1978l 1019-1025. 9 Hyyppa, M. T., Hyppothalamic monoamines in human fetuses, Neuroendocr., 9 (1972) 257-266. 10 lffy, L., Jakobovits, A., Westlake, W., Wingate, M., Caterin, H., Kanafsky, P. and Menduke, M., Early intrauterine development, Pediatrics, 56 (1975) 173-186.
oestradiol assays; Miss G. D o c h e r t y a n d Miss Alison M c C u l l o c h for their excellent technical assistance a n d Mrs M o i r a M c R a e for typing this m a n u s cript. This work was supported by a g r a n t to N . A . M . from the British Council, L o n d o n , u n d e r the Technical C o - o p e r a t i o n T r a i n i n g D e v e l o p m e n t P r o g r a m m e of the C o l o m b o Plan.
I l Laverty, R. and Taylor, K. M., The fluorometric assay of catecholamines and related compounds, Analyt. Biochem., 22 (1968) 269-n279. 12 Nobin, A. and Bj6rklund, A., Topography of the monoamine neurone systems in the human brain as revealed in fetuses, Acta physiol, scand., 88, Suppl. 388 (1973) 1-40. 13 Palkovits, M., Brownstein, M., Saavedra, J. M. and Axelrod, J., Norepinephrine and dopamine content of hypothatamic nuclei of the rat, Brain Res., 77 (1974~ 137-149. 14 Reyes, F. !., Boroditsky, R. S., Winter, J. S. D. and Faiman, C., Studies on human sexual development. II. Fetal and maternal serum gonadotrophin and sex steroid concentratrions, J. Clin. Endocr. Metab., 38 (19741 612-617. 15 Reznikov, A. G., Nosenko, N. D. and Demkiv, L. P., New evidences for participation of biogenic mono-amines in androgen-dependent sexual differentiation of hypothalamic control of gonadotrophin secretion in rats, Endokrinologie, 73 (1979) 11-19. 16 Saavedra, J. M., Microquantitation of neurotransmitters in specific areas of the central nervous system. Int. Rev. NeurobioL. 21 (1979/ 259-274. 17 Smith, J. E.. Lane, J. D., Shea. P. A.. McBride, W. J. and Aprison, M. H., A method for concurrent measurement of picomole quantities of acetylcholine, dopamine, noradrenaline, serotonin, 5-hydroxytryptophan, 5-hydroxyindole acetic acid, tryptophan, tyrosine, glycine, aspartate, glutamate, alanine and gamma aminobutyric acid in single tissue samples from different areas of rat central nervous system, Analyt. Biochem.. 64 (1975) 149-169. 18 Snyder, S. H., Axelrod, J. and Zweig, M., A sensitive and specific fluorescence assay for tissue serotonin, Biochem. Pharmacot., 14 (1965) 831-835. 19 Udenfriend, S., Weissbach, H. and Brodie, B. B., Assay of serotonin and related metabotites, enzymes and drugs, Meth. Bochern. Analysis', 6 (1958) 95-130. 20 Young, S. N.. Anderson, G. M., Gauthier, S. and Purdy, W. C., The origin of indoleacetic acid and indolepropionic acid in rat and human cerebrospinal fluid, J. Neurochem.. 34 (1980) 1087-1092.
9
Biogenic Amine Levels in the Mid-Term Human Fetus NAYYAR A. MASUDI* and DESMOND P. GILMORE** Institute of Physiology, The University, Glasgow G12 8QQ, Scotland ( U.K.) (Accepted June 29th, 1982) Key words: fetal brain - - CSF - - biogenic amine concentrations
Spectrophotofluorometric assays were used to measure concentrations of DA, NA, 5-HT and 5-HIAA present in extracts of brain tissue and CSF taken from midterm gestational fetuses of both sexes. The presence of biogenic amines was demonstrated in fetuses as early as 10.5 weeks gestation. In the hypothalamus, the concentrations (expressed in ng/100 mg tissue) ranged from 10.2 to 62.2 for NA, 11.6 to 258.8 for DA, 7.6 to 38.6 for 5-HT and 56.6 to 114.1 for 5-HIAA. Concentrations of DA and 5-HT were found to be significantly(P < 0.05) higher in the hypothalamus than in the cortex, but those of NA and 5-HIAA were similar in both areas. No sex differencewas observed. The concentrations of biogenicamines in the CSF (expressed in ng/ml) were 63.1-286.7 for NA, 43.2-108.1 for DA, 30.9-87.5 for 5-HT and 8.0-42.8 for 5-HIAA. INTRODUCTION It has recently been shown 15 that the catecholamines and serotonin (5-HT) play a role in the androgen-dependent sexual differentiation of the hypothalamus in rats and it is possible that in the human too, they serve a similar function. However, little knowledge at present exists about the concentrations of the different biogenic amines in the brain during human fetal development. In the present study concentrations of noradrenaline (NA), dopamine (DA), 5-HT and 5-hydroxyindole acetic acid (5-HIAA) were measured in the brain and cerebrospinal fluid (CSF) of mid-term human fetuses. MATERIALS AND METHODS Human fetuses were collected immediately after their removal from the uterus by hysterotomy (performed for gynaecosocial reasons under general anaesthesia). They were packed in ice and brought to the Institute of Physiology, where dissection was carried out. The time on average between hystero-
tomy termination and freezing of the tissue in liquid nitrogen was less than 45 min. Fetal sex was initially determined by examination of the external genitalia and subsequently confirmed by histological examination o f the gonads. Cord serum was collected whenever possible, and CSF was aspirated by lumbar puncture. These were frozen in liquid nitrogen and stored at - - 2 0 °C. Crown-rump length was measured and the fetuses were weighed. These measurements were compared with those in the Table published by Iffy et alA ° for age assessment. The brain was exposed by first making an incision across the frontal bones of the skull. The hypothalamus and a piece of cortical tissue from the frontal lobe were removed and separately frozen in liquid nitrogen. These samples were also stored at --20 °C until assayed. The material examined in this study included the hypothalamus and a piece of frontal lobe from 33 fetal brains, 5 whole brains (from smaller fetuses where removal of the hypothalamus was not possible), and CSF samples from 16 fetuses of midterm gestational age. Cord serum was obtained from
* Present address: Physiology Department, Sind Medical College, Jinnah Postgraduate Medical Centre, J.P.M.C. Campus, Karachi-35, Pakistan. ** To whom correspondence and reprint requests should be addressed. 0165-3806/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
10 a total of 32 male and 21 female fetuses and the testosterone and oestradiol in it measured by radioimmunoassay according to the procedure outlined by Cook et al. 6. Weighed pieces of brain were • ' / allowed to thaw and then homogentzed m 0.5 O,% HC1 following a similar procedure to that described by Campbell et al. 4. The final volume of the extracts was adjusted so that 1.0 ml of extract was equivalent to 160 mg of brain tissue. In these 1.0 ml brain tissue extracts, and in 1.0 ml CSF samples, NA and DA concentrations were measured using slight modifications of the method described by Laverty and Taylor 11. The procedure followed by Snyder et al. t8 was used to estimate concentrations of 5-HT in the samples and that of Udenfrend et al. 19 for 5-HIAA. A small number of extracts and CSF samples were purified by being chromatographed on a Bio-Gel P~ column (90 × 1.5 cm). The gel used had a 200-400 mesh size. The eluant buffer was Na H~PO4 (0.05 M, pH 7.5). H u m a n serum albumin was added to the eluarU to prevent non-specific binding to the gel or to the glass of the column. The flow rate was 6 ml/h and the column was run at a temperature of 4 °C. A total of 200 1.0 ml fractions were collected from each column run. RESULTS The results of the non-eluted extracts and CSF samples are summarized in Table I and those eluted on the Bio-Gel P2 column in Table II. Values (corrected for recovery) recorded for the different amines are expressed in ng/100 mg wet weight of brain tissue and ng/ml of CSF. Differences were analyzed statistically by Student's t-test. F r o m the non-eluted extracts and CSF samples
recoveries of internal standards were 70'~,i for NA, 68'~o for DA, 65 !V,,for 5-HT and 75'%~I for 5-HIAA. It can be seen that although there are significantly higher levels (P < 0.05) of DA and 5-HT in the hypothalamus compared to the cortex, the hypothalamic and cortical concentrations of" NA were not significantly different. Concentrations of 5H I A A in the cortex and hypothalamus were also similar. Concentrations of NA. DA, 5-HT and 5H I A A in the CSF of mid-term fetuses showed no significant sex differences. The concentration of 5HIAA in the human fetal CSF found in this study (19. I :k 3.2 ng/ml) was in agreemenl with a previous report 20 which showed that 5-HIAA concentrations in the adult human lumbar CSF were 22.0 ! t2.4 ng/ml and in the human cisternal CSF 27.7 i- 13.0 ng/ml. Concentrations of biogenic amines covering the area of each peak identified by its native fluorescence in the eluted fractions of human fetal brain tissue extractions and CSF samples from the BioGel Pz columns are presented in Table 1I. Recoveries of internal standards for NA, DA and 5-HT in the eluted fractions of hypothalamic extracts were () ( ) ,' 81.3y,',, 63.3/~, and 63.00/o respectively. Internal standard recoveries in the eluted fractions of whole brain extracts were 83 ~ for NA, 95 "~ for D A and 60 °j for 5-HT, while recoveries of" the internal /o standards in the CSF samples were 60°'~ for NA, 81 o~ for DA and 73 ~,/, for 5-HT. No significant differences in NA, DA and 5-HT concentrations (Table lI) were observed between the whole brain tissue hypothalamic and CSF samples run through the Bio-Get P2 columns, although their presence was again quantitatively demonstrated.
TABLE 1 Concentrations of NA, DA, 5-HT and 5-HIAA in the mid-term fetus ( ~ ± S.E.M.)
Cortex(ng/10Omg) Range Hypothalamus(ng/10Omg) Range CSF (ng/ml) Range
NA
DA
5-HT
5-HIAA
35.1 ~+_ 5.1 (19) (6.8-79.2) 34.4 ± 3.0 (21) (10.2-62.2) 143.8 3- 20.9 (11) (63 •1-286.7)
73.t 3_ 7.6 (17) (18.0-123.9) 118.1 ~ 16.9 (21)* (11.6-258.8) 70.4 L 5.6 (11) (43.2-108.1)
14.0 -t: 1.8 (17) (3.2-31.2) 21.0 ~ 3.1 (12)* (7.6-38.6) 60.2 :~ 4.8 (1l) (30.9-87.5)
75.9 :!- 4.0 (12) (48.5--95.5) 79.0 4.8 (12) (56.6-114.1 ) 19.1 3.2(11) (8.0:-42.8)
* Significantly higher than in cortex, P • 0.05.
11 TABLE II Concentrations of biogenic amines in the eluted fractions of human fetal brain tissue extracts run through Bin-Gel P~ columns ( ~ ± S.E.M.)
Whole brain (ng/100 mg) Hypothalamus (ng/100 mg) CSF (ng/ml)
?CA
DA
5-HT
151.0 ± 6.8 (5) 126.2 ± 12.8 (5) 175.4 ± 7.3 (4)
141.3 ± 14.8 (5) 131.4 ± 15.9 (5) 58.1 ± 7.4 (4)
33.5 ± 3.6 (5) 31.0 ± 3.1 (5) 38.7 ± 4.6 (4)
The radioimmunoassay for testosterone showed an intraassay variation of 9 ~ and an interassay variation of 12 ~. The variations for oestradiol were 14 ~ and 17 ~ respectively. Levels of oestradiol and testosterone in the cord serum were very similar to those recorded by other workers 14. These were 4.9 :k 0.6 ng/ml (n --~ 32) in the male and 1.2 4- 0.2 (n ---- 21) in the female. The higher serum testosterone values, up to 16.7 ng/ml, were seen in fetuses between 11 and 18 weeks of age; after this time testosterone levels declined reflecting the falling levels of hCG and thus the reduced stimulus to the interstitial Leydig cells. Human fetal oestradiol concentrations showed no significant sex difference, being 1.7 ± 0.3 ng/ml (n = 13) in the male and 1.8 4- 0.4 ng/ml (n ~ 10) in the female. DISCUSSION The spectrophotofluorometric methods used in this study were able to demonstrate clearly the presence of biogenic amines in the mid-term fetus and also measure their concentrations in the different brain tissues and CSF. The methods used were found to be both sensitive and accurate and could detect biogenic amine concentrations of as low as 1-10 ng/100 mg tissue. Observations made in this study upon the hypothalamic concentrations of catecholamines and indoleamines are from the whole hypothalamus and not from individual nuclei. In contrast, much recent work has been carried out on the rat and centered upon the concentrations of DA, NA, 5-HT and 5-HIAA in specific regions of the hypothalamus and braina,S,la,16,aT. In primates the concentrations of DA, N A , 5-HT and 5-HIAA in the hypothalamus and other parts of the rhesus monkey brain have been also reported 2. More recently the levels of these substances were also in-
vest±gated in the adult human brain 5. In the present study the presence of biogenic amines was detected and measured in fetuses of as early as 10.5 weeks gestational age confirming the observations of a previous finding 9. The concentration of NA in the whole human fetal brain (10.5-13.5 weeks gestational age) was found to be 209 ng/100 mg of tissue while that of DA was 106 ng/100 mg. The ratio of NA/DA was 1.98. This agrees closely with NA/DA ratio of 2 measured in the brain of a single 20-week-old fetus 1. The whole brain catecholamine levels in 3 fetuses of 12-16 weeks gestational age were determined and found to be very low 12. A mean of 244 ng of NA and 101 ng of DA was found per brain, corresponding to a concentration of only 18 ng and 9 ng/g brain tissue respectively12. However, the NA/DA ratio was 2 which is again in good agreement with the pattern obtained in this study. In the hypothalamic extracts the NA and DA concentrations were found, as might be expected, to be different from those in the brain extracts. In the human fetal hypothalamus NA levels were 34 ng/100 mg of tissue while DA levels were 118 ng/100 mg of tissue. The NA/DA ratio here was 0.3. This is not dissimilar to the ratio of 0.46 for the rat fetal whole brain of 17 days gestation 7. One striking feature resulting from the present investigation was that 5-HT concentrations in the human fetal brain tissue were found to be higher than those previously described for the adult. The concentrations of 5-HT in the adult human hypothalamus was 10 ng/100 mg and in the cortex 1.05 ng/100 mg tissue 5. This contrasts with the values of 21 ng/100 mg in the fetal hypothalamus and 35 ng/100 mg in the fetal cortex recorded in the present study. The concentrations of 5-HIAA found in the hypothalamus (79 ng/100 rag) and cortex (73 ng/100 rag)
12 of h u m a n fetuses were very similar a n d n o t significantly different f r o m the values reported for the
ACKNOWLEDGEMENTS
adult rat h y p o t h a l a m u s a n d b r a i n 8,17. The concen-
We t h a n k Professor M. C. M a c N a u g h t o n , Dr.
trations of 5 - H I A A in the h u m a n fetal C S F samples
A n d r e w Calder a n d their staff at Glasgow Royal
(19 ng/ml) recorded in the present study, were also
Infirmary for providing hysterotomy specimens; Dr. Brian Cook for carrying out testosterone a n d
similar to those reported for adult h u m a n l u m b a r C S F values (22 ng/ml). The results of the present study for 5 - H I A A c o n c e n t r a t i o n s are also in good agreement with the findings of Brown et al. e, who showed a similar p a t t e r n of 5 - H I A A c o n c e n t r a t i o n s in the m o n k e y brain. Moreover, the presence of 5H I A A in the h u m a n fetuses clearly indicates the rapid b r e a k d o w n of 5-HT in the b r a i n tissue.
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oestradiol assays; Miss G. D o c h e r t y a n d Miss Alison M c C u l l o c h for their excellent technical assistance a n d Mrs M o i r a M c R a e for typing this m a n u s cript. This work was supported by a g r a n t to N . A . M . from the British Council, L o n d o n , u n d e r the Technical C o - o p e r a t i o n T r a i n i n g D e v e l o p m e n t P r o g r a m m e of the C o l o m b o Plan.
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