Life Sciences Vol. 18, pp. 919-924 Printed in the U.S.A.
Pergamon Press
PLATELET MONOAMINE OXIDASE ACTIVITY IN CHILDREN AND ADOLESCENTS Jerome A. Roth I, J. Gerald Young, and Donald J. Cohen Department of Anesthesiology. 1 , Pediatrics, Psychiatry and Psychology Yale University School of Medicine 333 Cedar Street New Haven, Connecticut
06510
(Received in final form March 15, 1976) SUMMARY Platelet monom, ine oxidase activity has been measured in a group of preadolescent, adolescent, and post-adolescent individuals. The platelet oxidase activity was highest in the female in all three populations examined. Children diagnosed as having primary autism did not display differences in their plateletoxldase activity as compared to children of similar age and sex. Human blood platelet monoamine oxldase (MAO) has been used as a peripheral marker for the activity of this enzyme in brain (1,2). Though the human platelet oxidase has been reported to be homogeneous based on acrylamide gel electrophoresis and to have properties of the B type of MAO, recent studies have indicated that this enzyme may be comprised of several isoenzymes or contain multiple binding sites (3,4). Activity of the platelet oxidase is influenced by a number of factors including age (1,5), sex (1,2), hormones (6), nutrition (7,8), drugs (9) and the enzyme appears to be genetically controlled with a variance of about 17~ which is noninheritable (2). Several recent papers have indicated that platelet MAO activity is reduced in the chronic schizophrenic (10,11) and bipolar depressed patient (12), and is elevated in a mixed group of depressed patients (13). There was no difference in the activity of this oxldase in the acute schizophrenic as compared to that of a normal population (14). The information described above has largely been obtained with an adult population over the age of 20 and there is no information in the literature which describes the activity of this enzyme in adolescent or preadolescent children. Since the variation in platelet MAO activity between males and females is generally attributed to hormonal differences in the sexes, in this paper the activity of this enzyme was assessed in a group of normal children to determine whether these differences also exist prior to and during puberty and to measure the oxidase activity after the onset of puberty when hormonal changes are the greatest. METHODS Blood samples (7 ml) were collected in glass tubes containing 10.5 EDTA (K3) (Becton, Dickinson & Co. #4675). Normally, a total of 14 ml blood was collected from each subject. Blood samples were immediately in ice and were centrifuged in succession at 175 x g and 300 x g, each 919
mg of placed for I0
920
Human Platelet MAO
Vol. 18, No. 9
minutes, witllin a 3 hour period after collection. The resulting platelet rich plasma was centrifuged at 4,000 x g for 20 minutes to precipitate the platelets. The platelet pellet was resuspended in 2 ml of cold isotonic saline and recentrifuged at i0,000 x g for an additional 20 minutes. The saline was discarded and the platelets were stored frozen. All steps subsequent to collection were done at 0-4 ° using plastic pipets and containers. The frozen platelets were resuspended in 2 ml of 0.i M potassium phosphate buffer, pH 7.4, and sonicated three times at 30 sec. intervals. Platelet MAO was measured by a modification of the procedure described by Roth and Gillis (15). In brief, reaction mixtures containing 0.5 ml of the platelet suspension were incubated with 1.67 x 10 -4 M 14C-tyramine in a total volume of 1.5 ml of 0.05 M potassium phosphate buffer, pH 7.4. In all cases less than 15% metabolism of the labelled tyramine occurred. The concentration of tyramine used was approximately 5 fold greater than its reported K m value of 3.11 x 10 -5 M (3). Reaction mixtures were incubated for 60 minutes at 37 ° , and terminated by addition of 0.2 ml of 0.25 M zinc sulfate followed by addition of 0.2 ml of a saturated solution of barium hydroxide. The resulting precipitate was removed by centrifugation and 0.5 ml aliquots of the supernatant solutions were passed through Bio Rex-70 cation exchange resin (Sodium form; 100-200 mesh; equilibrated with 0.4 M sodium phosphate buffer, pH 6.0) columns. The deaminated products formed in the reaction were removed from the resin with 2.5 ml of water which was combined with the 0.5 ml eluate above. Ten ml of Aquasol was added to the total eluate and the total radioactivity was measured in a Packard scintillation spectrometer (Model 3320). All assays were performed in duplicate. Protein concentrations were determined by the method of Lowry et al. (16). RESULTS The reproducibility of the assay was tested by determining MAO activity in platelets derived from i0 children undergoing an ~nsulin tolerance test. Blood samples were obtained from each child prior to, 60 minutes after and, also, in some cases 90 minutes after beginning the insulin infusion. The mean percent difference for the MAO activity for each child was determined to be 5.0%. The variability of platelet MAO activity was also estimated in i0 individuals, 6 females and 4 males, over a period of several months. The average percent difference in the platelet oxidase activity for each subject was found to be 17.7%. This value is similar to that obtained by Murphy e__~t al. (2) for percent difference in MAO activity from individuals whose bloods were drawn one week apart. We found that variability in females was considerably greater than in the male, 30% versus 10%, respectively. The effect of age and sex on platelet MAO activity is shown on the bar graph in Figure i. As can be seen there was no increase in the oxidase activity within the age groups examined. This was verified by the fact that correlation coefficients determined for graphs of age versus MAO activity for either male or female were not significantly different, -0.295 and - 0.057, respectively. It should be pointed out that the increase in MAO activity reported by others (1,5) is not usually observed until the age of 50. The oldest male we examined was 51 and the oldest female was 48. The data in Figure 1 also demonstrates that the activity of this enzyme is greater in the female than in the male at all age groups examined except between the ages of 20 to 30. Of the three females examined between the ages of 20 to 30, one had taken 30 mg of valium three-and-one-half hours prior to her blood sampling. Her MAO activity, 2.85 nmoles of deaminated product formed per mg of protein, was the lowest of the entire female population tested.
Vol. 18, No. 9
H,-,-n Platelet MA0
921
~'~ Mole
-
A
48
~
32
16
8
Fomole
i i l'ii
0-5
5-10
10-15 15-20 20-30 30-40
>40
AGE
FIG. 1 Platelet MAO activity was assayed at 37 ° with 1.67 x 10 -4 M 14C-tyrsmine as substrate in a total of 1.5 ml of 0.05 M potassium phosphate buffer, pH 7.4. Activity is expressed as nmoles deaminated product formed/mg protein/ 60 m/n. incubation + S.E. Numbers on top of bars represent the sample population. The mean value for MAO activity in male and female platelets prior to, during and after puberty, as well as in the entire population examined is presented in Table I. MAO activity is greater in the female than in the male in all three subpopulatlons listed. Robinson et al. (I) using benzylamine as substrate reports that in the adult female, MAO activity is approximately 27% greater than that of the male. We obtained a similar value for percent increase in platelet oxidase activity, 23%, for a similar female population. Values of 52% and 61% were obtained for the difference in MAO activity for the populations prior to and during puberty, respectively. This larger difference in male-female MAO activity in children reflects the fact that there is also a significant decrease in the activity of this oxldase in females after puberty (age > 15). The platelet deaminating activity in males remains constant over the entire three subgroupings. The decrease in the activity in the females is still significant even if the value for the subject who took 30 mg of valium is omitted. One of the primary aims of this study was to determine the platelet MAO activity of 33 children (30 males and 3 females) afflicted with primary childhood autism (Kanner's syndrome). The diagnostic criteria have previously been reported (17). The mean value for nmoles d e a ~ n a t e d product formed per mg of platelet protein in male and female autistic children was 25.22 + 1.38 and 32.39 ~ 2.54, respectively, and was not significantly different from that of the normal population. In addition to these studies we also measured the MAO activity of members
922
Human Platelet MAO
In .
c
Vol. 18, No. 9
Vol. 18, No. 9
Human Platelet MAO
923
of eleven familles. An analysls of variance of this group of families revealed that the variance in the oxldase activity within a given family was significantly smaller than that between the families (F - 3.2; P < 0.01). These results are consistent with prevlously published reports which indicate that platelet MAO activity is genetlcally controlled (18, 19). DISCUSSION Unpublished observations in this laboratory indicate that the human brain type B MAO possesses similar substrate and inhibitor specificity as that of the platelet oxldase. If the activity of platelet MAO actually represents that in brain, it Is interesting to find that females have a higher level of activity than the male even before puberty. Though it is generally assumed that the difference In this activity in adult male and female results from hormonal differences, it is dlfflcult to explain our observation with prepubescent children since it has been shown that the levels of the steroid hormones between the sexes prior to puberty are not appreciably different (20-22). In addition there is no marked change in the level of the oxldase at the onset of puberty at which time hormonal changes are greatest. Results In this paper are consistent with the premise that the platelet oxldase Is genetically controlled. Platelet MAO activity has been reported to be reduced In the chronic schizophrenic and because MAO activity is similar in twins discordant for schizophrenia, it has been suggested that platelet activity may be used as a genetic marker for this disease (10,11). However, there was no difference in the activity of this oxldase in children diagnosed as having chlldhood autism. Further studies are presently being conducted wlth children suffering from other severe developmental disorders in order to determine whether their platelet MAO activity is altered. ACKNOWLEDGEMENTS We thank Ms. Mary Ellen Kavanagh for her excellent technical and clinical assistance; Drs. R. Feiz, C. Rich, and R. Wlseman, and the staff of the Children's Unit, Connecticut Valley Hospital; MS. Barbara Caparulo and Mr. Warren Johnson for assistance In research coordination end patient care; and the staff of the Children's Clinical Research Center, under the direction of Dr. M. Genel and Mary Carey, R.N. This paper was supported in part by Public Health Service Research Grant DH-03008; the Grant Foundation of New York; the Children's Clinical Research Grant RR 00125, General Clinical Research Centers Program, Division of Research Resources, National Institutes of Health; National Research Service Award MH 05223, National Institute of Mental Health; National Institute of Heart and Lung Grant HL 17124; National Institute of Mental Health Grant MH 26354; and a grant from Mr. Leonard Berger. Present address of Dr. J. A. Both is Department of Pharmacology and Therapeutics, School of Medicine and Dentistry, State University of New York at Buffalo, Buffalo, New York 14214. REFERENCES i. 2.
D.S. ROBINSON, J.M. DAVIS, A. NIES, C.L. RAVARIS & D. SYLWESTER, Arch. Gen. Psychlat.,24, 536-539 (1971). D.L. MURPHY, R. B MAKER, & R.J. WYATT, J. Psychlat. Res., ii, 221-247
3.
G.G.S. COLLINS & M. SARDEER, Biochem. Pharmacol., 20, 289-296 (1971).
4.
D.J. EDWARDS & S.S. C~NG, Btochem. Btophys. Res.. Co~,n., 65, 1018-1025 (1975).
(1974).
924
5. 6. 7. 8. 9. i0. ii. 12. 13.
14. 15. 16. 17. 18. 19. 20. 21. 22.
Human Platelet MAO
Vol. 18, No. 9
D.S. ROBINSON, Fed. Proc., 34, 103-107 (1975). R.H. BELMAKER, D.L. MURPHY, R.J. WYATT & R.J. LORIAUX, Arch. Gen. Psychiat., 31, 553-556 (1974). M.B.H. YOUDIM & T.L. SOURKES, Can. J. Biochem., 43, 1305-1318 (1965). M.B. YOUDIM, H.F. WOODS, B. MITCHELL, D.C. GRAHAME-SMITH & S. CALLENDER, Clin. Sci. Molec. Med., 48, 289-295 (1975). D.S. ROBINSON, W. LOVENBERG, H. KEISER, & A. SZOERDSMA, Biochem. Pharmacol, 17, 109-119 (1968). D.L. MIJRPHY & R.J. WYATT, Nature, 238, 225-226 (1972). H.Y. MELTZER & S.M. STAHL, Res. Comm. Chem. Pathol. Pharmacol., 7, 419431 (1974). D.L. MURPHY & R. WEISS, Am. J. Psychiat., 128, 1351-1357 (1972). A. NIES, D.S. ROBINSON, L.S. HARRIS & K.R. LAMBORN, in Neuropsychopharmacology of Monoamines and Their Regulatory Enzymes (ed. by Usdin, E.) 5970 (Raven Press, New York, 1974). W.T. CARPENTER, D.L. MURPHY & R.J. WYATT, Arch. Gen. Psychiat., 132, 438441 (1975). J.A. ROTH & C.N. GILLIS, Molec. Pharmacol., ii, 28-35 (1975). O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR & R.J. RANDALL, J. Biol. Chem., 193, 265-275 (1951). D.J. COHEN, B.A.SHAYWITZ, W.T. JOHNSON & M. BOWERS, JR., Arch. Gen. Psychiat., 31, 845-853 (1974). A. NIES, D.S. ROBINSON, K.R. LAMBORN & R.P. LAMPERT, Arch. Gen. Psychiat., 28, 834-838 (1973). R.J. WYATT, D.L. MURPHY, R. BELMAKER, S. COHEN, C.H. DONNELLY & W. POLLIN, Science, 179, 916-918 (1973). A.W. ROOT, J. Pediat., 83, 1-19 (1973). M. GRUMBACH, G. GRAWE & F. MAYER, in The Control of the Onset of Puberty (John Wiley and Sons, New York, 1974). D. COHEN & R. FRANK, in Mental Health in Children: Psychiatric and Psychological Problem_~s (ed. Sankar, D.V.S.) (P.J.D. Publications, Ltd., Westbury, New York, 1975).
Pergamon Press
PLATELET MONOAMINE OXIDASE ACTIVITY IN CHILDREN AND ADOLESCENTS Jerome A. Roth I, J. Gerald Young, and Donald J. Cohen Department of Anesthesiology. 1 , Pediatrics, Psychiatry and Psychology Yale University School of Medicine 333 Cedar Street New Haven, Connecticut
06510
(Received in final form March 15, 1976) SUMMARY Platelet monom, ine oxidase activity has been measured in a group of preadolescent, adolescent, and post-adolescent individuals. The platelet oxidase activity was highest in the female in all three populations examined. Children diagnosed as having primary autism did not display differences in their plateletoxldase activity as compared to children of similar age and sex. Human blood platelet monoamine oxldase (MAO) has been used as a peripheral marker for the activity of this enzyme in brain (1,2). Though the human platelet oxidase has been reported to be homogeneous based on acrylamide gel electrophoresis and to have properties of the B type of MAO, recent studies have indicated that this enzyme may be comprised of several isoenzymes or contain multiple binding sites (3,4). Activity of the platelet oxidase is influenced by a number of factors including age (1,5), sex (1,2), hormones (6), nutrition (7,8), drugs (9) and the enzyme appears to be genetically controlled with a variance of about 17~ which is noninheritable (2). Several recent papers have indicated that platelet MAO activity is reduced in the chronic schizophrenic (10,11) and bipolar depressed patient (12), and is elevated in a mixed group of depressed patients (13). There was no difference in the activity of this oxldase in the acute schizophrenic as compared to that of a normal population (14). The information described above has largely been obtained with an adult population over the age of 20 and there is no information in the literature which describes the activity of this enzyme in adolescent or preadolescent children. Since the variation in platelet MAO activity between males and females is generally attributed to hormonal differences in the sexes, in this paper the activity of this enzyme was assessed in a group of normal children to determine whether these differences also exist prior to and during puberty and to measure the oxidase activity after the onset of puberty when hormonal changes are the greatest. METHODS Blood samples (7 ml) were collected in glass tubes containing 10.5 EDTA (K3) (Becton, Dickinson & Co. #4675). Normally, a total of 14 ml blood was collected from each subject. Blood samples were immediately in ice and were centrifuged in succession at 175 x g and 300 x g, each 919
mg of placed for I0
920
Human Platelet MAO
Vol. 18, No. 9
minutes, witllin a 3 hour period after collection. The resulting platelet rich plasma was centrifuged at 4,000 x g for 20 minutes to precipitate the platelets. The platelet pellet was resuspended in 2 ml of cold isotonic saline and recentrifuged at i0,000 x g for an additional 20 minutes. The saline was discarded and the platelets were stored frozen. All steps subsequent to collection were done at 0-4 ° using plastic pipets and containers. The frozen platelets were resuspended in 2 ml of 0.i M potassium phosphate buffer, pH 7.4, and sonicated three times at 30 sec. intervals. Platelet MAO was measured by a modification of the procedure described by Roth and Gillis (15). In brief, reaction mixtures containing 0.5 ml of the platelet suspension were incubated with 1.67 x 10 -4 M 14C-tyramine in a total volume of 1.5 ml of 0.05 M potassium phosphate buffer, pH 7.4. In all cases less than 15% metabolism of the labelled tyramine occurred. The concentration of tyramine used was approximately 5 fold greater than its reported K m value of 3.11 x 10 -5 M (3). Reaction mixtures were incubated for 60 minutes at 37 ° , and terminated by addition of 0.2 ml of 0.25 M zinc sulfate followed by addition of 0.2 ml of a saturated solution of barium hydroxide. The resulting precipitate was removed by centrifugation and 0.5 ml aliquots of the supernatant solutions were passed through Bio Rex-70 cation exchange resin (Sodium form; 100-200 mesh; equilibrated with 0.4 M sodium phosphate buffer, pH 6.0) columns. The deaminated products formed in the reaction were removed from the resin with 2.5 ml of water which was combined with the 0.5 ml eluate above. Ten ml of Aquasol was added to the total eluate and the total radioactivity was measured in a Packard scintillation spectrometer (Model 3320). All assays were performed in duplicate. Protein concentrations were determined by the method of Lowry et al. (16). RESULTS The reproducibility of the assay was tested by determining MAO activity in platelets derived from i0 children undergoing an ~nsulin tolerance test. Blood samples were obtained from each child prior to, 60 minutes after and, also, in some cases 90 minutes after beginning the insulin infusion. The mean percent difference for the MAO activity for each child was determined to be 5.0%. The variability of platelet MAO activity was also estimated in i0 individuals, 6 females and 4 males, over a period of several months. The average percent difference in the platelet oxidase activity for each subject was found to be 17.7%. This value is similar to that obtained by Murphy e__~t al. (2) for percent difference in MAO activity from individuals whose bloods were drawn one week apart. We found that variability in females was considerably greater than in the male, 30% versus 10%, respectively. The effect of age and sex on platelet MAO activity is shown on the bar graph in Figure i. As can be seen there was no increase in the oxidase activity within the age groups examined. This was verified by the fact that correlation coefficients determined for graphs of age versus MAO activity for either male or female were not significantly different, -0.295 and - 0.057, respectively. It should be pointed out that the increase in MAO activity reported by others (1,5) is not usually observed until the age of 50. The oldest male we examined was 51 and the oldest female was 48. The data in Figure 1 also demonstrates that the activity of this enzyme is greater in the female than in the male at all age groups examined except between the ages of 20 to 30. Of the three females examined between the ages of 20 to 30, one had taken 30 mg of valium three-and-one-half hours prior to her blood sampling. Her MAO activity, 2.85 nmoles of deaminated product formed per mg of protein, was the lowest of the entire female population tested.
Vol. 18, No. 9
H,-,-n Platelet MA0
921
~'~ Mole
-
A
48
~
32
16
8
Fomole
i i l'ii
0-5
5-10
10-15 15-20 20-30 30-40
>40
AGE
FIG. 1 Platelet MAO activity was assayed at 37 ° with 1.67 x 10 -4 M 14C-tyrsmine as substrate in a total of 1.5 ml of 0.05 M potassium phosphate buffer, pH 7.4. Activity is expressed as nmoles deaminated product formed/mg protein/ 60 m/n. incubation + S.E. Numbers on top of bars represent the sample population. The mean value for MAO activity in male and female platelets prior to, during and after puberty, as well as in the entire population examined is presented in Table I. MAO activity is greater in the female than in the male in all three subpopulatlons listed. Robinson et al. (I) using benzylamine as substrate reports that in the adult female, MAO activity is approximately 27% greater than that of the male. We obtained a similar value for percent increase in platelet oxidase activity, 23%, for a similar female population. Values of 52% and 61% were obtained for the difference in MAO activity for the populations prior to and during puberty, respectively. This larger difference in male-female MAO activity in children reflects the fact that there is also a significant decrease in the activity of this oxldase in females after puberty (age > 15). The platelet deaminating activity in males remains constant over the entire three subgroupings. The decrease in the activity in the females is still significant even if the value for the subject who took 30 mg of valium is omitted. One of the primary aims of this study was to determine the platelet MAO activity of 33 children (30 males and 3 females) afflicted with primary childhood autism (Kanner's syndrome). The diagnostic criteria have previously been reported (17). The mean value for nmoles d e a ~ n a t e d product formed per mg of platelet protein in male and female autistic children was 25.22 + 1.38 and 32.39 ~ 2.54, respectively, and was not significantly different from that of the normal population. In addition to these studies we also measured the MAO activity of members
922
Human Platelet MAO
In .
c
Vol. 18, No. 9
Vol. 18, No. 9
Human Platelet MAO
923
of eleven familles. An analysls of variance of this group of families revealed that the variance in the oxldase activity within a given family was significantly smaller than that between the families (F - 3.2; P < 0.01). These results are consistent with prevlously published reports which indicate that platelet MAO activity is genetlcally controlled (18, 19). DISCUSSION Unpublished observations in this laboratory indicate that the human brain type B MAO possesses similar substrate and inhibitor specificity as that of the platelet oxldase. If the activity of platelet MAO actually represents that in brain, it Is interesting to find that females have a higher level of activity than the male even before puberty. Though it is generally assumed that the difference In this activity in adult male and female results from hormonal differences, it is dlfflcult to explain our observation with prepubescent children since it has been shown that the levels of the steroid hormones between the sexes prior to puberty are not appreciably different (20-22). In addition there is no marked change in the level of the oxldase at the onset of puberty at which time hormonal changes are greatest. Results In this paper are consistent with the premise that the platelet oxldase Is genetically controlled. Platelet MAO activity has been reported to be reduced In the chronic schizophrenic and because MAO activity is similar in twins discordant for schizophrenia, it has been suggested that platelet activity may be used as a genetic marker for this disease (10,11). However, there was no difference in the activity of this oxldase in children diagnosed as having chlldhood autism. Further studies are presently being conducted wlth children suffering from other severe developmental disorders in order to determine whether their platelet MAO activity is altered. ACKNOWLEDGEMENTS We thank Ms. Mary Ellen Kavanagh for her excellent technical and clinical assistance; Drs. R. Feiz, C. Rich, and R. Wlseman, and the staff of the Children's Unit, Connecticut Valley Hospital; MS. Barbara Caparulo and Mr. Warren Johnson for assistance In research coordination end patient care; and the staff of the Children's Clinical Research Center, under the direction of Dr. M. Genel and Mary Carey, R.N. This paper was supported in part by Public Health Service Research Grant DH-03008; the Grant Foundation of New York; the Children's Clinical Research Grant RR 00125, General Clinical Research Centers Program, Division of Research Resources, National Institutes of Health; National Research Service Award MH 05223, National Institute of Mental Health; National Institute of Heart and Lung Grant HL 17124; National Institute of Mental Health Grant MH 26354; and a grant from Mr. Leonard Berger. Present address of Dr. J. A. Both is Department of Pharmacology and Therapeutics, School of Medicine and Dentistry, State University of New York at Buffalo, Buffalo, New York 14214. REFERENCES i. 2.
D.S. ROBINSON, J.M. DAVIS, A. NIES, C.L. RAVARIS & D. SYLWESTER, Arch. Gen. Psychlat.,24, 536-539 (1971). D.L. MURPHY, R. B MAKER, & R.J. WYATT, J. Psychlat. Res., ii, 221-247
3.
G.G.S. COLLINS & M. SARDEER, Biochem. Pharmacol., 20, 289-296 (1971).
4.
D.J. EDWARDS & S.S. C~NG, Btochem. Btophys. Res.. Co~,n., 65, 1018-1025 (1975).
(1974).
924
5. 6. 7. 8. 9. i0. ii. 12. 13.
14. 15. 16. 17. 18. 19. 20. 21. 22.
Human Platelet MAO
Vol. 18, No. 9
D.S. ROBINSON, Fed. Proc., 34, 103-107 (1975). R.H. BELMAKER, D.L. MURPHY, R.J. WYATT & R.J. LORIAUX, Arch. Gen. Psychiat., 31, 553-556 (1974). M.B.H. YOUDIM & T.L. SOURKES, Can. J. Biochem., 43, 1305-1318 (1965). M.B. YOUDIM, H.F. WOODS, B. MITCHELL, D.C. GRAHAME-SMITH & S. CALLENDER, Clin. Sci. Molec. Med., 48, 289-295 (1975). D.S. ROBINSON, W. LOVENBERG, H. KEISER, & A. SZOERDSMA, Biochem. Pharmacol, 17, 109-119 (1968). D.L. MIJRPHY & R.J. WYATT, Nature, 238, 225-226 (1972). H.Y. MELTZER & S.M. STAHL, Res. Comm. Chem. Pathol. Pharmacol., 7, 419431 (1974). D.L. MURPHY & R. WEISS, Am. J. Psychiat., 128, 1351-1357 (1972). A. NIES, D.S. ROBINSON, L.S. HARRIS & K.R. LAMBORN, in Neuropsychopharmacology of Monoamines and Their Regulatory Enzymes (ed. by Usdin, E.) 5970 (Raven Press, New York, 1974). W.T. CARPENTER, D.L. MURPHY & R.J. WYATT, Arch. Gen. Psychiat., 132, 438441 (1975). J.A. ROTH & C.N. GILLIS, Molec. Pharmacol., ii, 28-35 (1975). O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR & R.J. RANDALL, J. Biol. Chem., 193, 265-275 (1951). D.J. COHEN, B.A.SHAYWITZ, W.T. JOHNSON & M. BOWERS, JR., Arch. Gen. Psychiat., 31, 845-853 (1974). A. NIES, D.S. ROBINSON, K.R. LAMBORN & R.P. LAMPERT, Arch. Gen. Psychiat., 28, 834-838 (1973). R.J. WYATT, D.L. MURPHY, R. BELMAKER, S. COHEN, C.H. DONNELLY & W. POLLIN, Science, 179, 916-918 (1973). A.W. ROOT, J. Pediat., 83, 1-19 (1973). M. GRUMBACH, G. GRAWE & F. MAYER, in The Control of the Onset of Puberty (John Wiley and Sons, New York, 1974). D. COHEN & R. FRANK, in Mental Health in Children: Psychiatric and Psychological Problem_~s (ed. Sankar, D.V.S.) (P.J.D. Publications, Ltd., Westbury, New York, 1975).
