Nurrrophorm~,‘,,luy,
0028-3908/79/0401-0423X02.00/0
Vol. IS. pp. 423 lo 426
0 Pergamon Press Ltd 1979. Printed in Great Britain
DOPAMINE-#I-HYDROXYLASE AND NOREPINEPHRINE IN HUMAN CEREBROSPINAL FLUID: EFFECTS OF MONOAMINE OXIDASE INHIBITORS PAULINE LERNER’, L. F. MAJOR*, D. L. MURPHY’, S. LIPPER’, C. R. LAKES and W. LOVENBERG~ ‘Biological Psychiatry Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20014, U.S.A. ‘Clinical Neuropharmacology Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20014, U.S.A. jLaboratory of Clinical Science, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20014, U.S.A. and 4Hypertension-Endocrine Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20014, U.S.A. (Accepted 17 October 1978)
Summary-The effect of the monoamine oxidase inhibitors, clorgyline and pargyline, on central noradrenergic function in depressed patients was assessed by measuring dopamine beta hydroxylase (DBH) and norepinephrine in cerebrospinal fluid (CSF). These drugs caused a significant decrease in DBH in CSF. This decrease is consistent with a reduction in the release of DBH into CSF as noradrenergic firing is slowed by the drugs. Levels of DBH and norepinephrine in CSF were highly correlated. The ratio of CSF norepinephrine to CSF DBH increased when the patients were treated with monoamine oxidase inhibitors, consistent with a drug-induced increase in brain norepinephrine.
Dopamine-fl-hydroxylase (DBH), the enzyme which converts dopamine to norepinephrine, has been used as a marker for noradrenergic neurons in animals (Kopin, Kaufman, Viveros, Jacobowitz, Lake, Ziegler, Lovenberg and Goodwin, 1976) and humans (Hartman, 1974). It is localized along with norepinephrine in noradrenergic storage vesicles (Slotkin and Edwards, 1973; Coyle and Kuhar, 1974); when norepinephrine is released from these vesicles by exocytosis, the soluble fraction of DBH in the vesicles is also released (Viveros, Argueros and Kirshner, 1968; Smith and Winkler, 1972). Human serum contains a significant amount of DBH, which presumably originates from the sympathetic nervous system (Weinshilboum and Axelrod, 1971). The amount of DBH in human serum is largely determined by genetic factors, but can be changed, to a limited extent, by stressing the sympathetic nervous system (Kopin et al., 1976). The small magnitude of the observed changes in serum DBH levels may be explained by the large pool of DBH in serum and the slow turnover of the enzyme. Serum norepinephrine, like serum DBH, probably derives from sympathetic neurons. Marked changes in serum norepinephrine are seen when the sympathetic nervous system is stressed acutely (Lake, Ziegler, Coleman and Kopin, 1977). It has been suggested that serum norepinephrine is useful as a marker for sympathetic activity (Lake, Ziegler and Kopin, 1976). Key words : dopamine beta hydroxylase, norepinephrine, cerebrospinal fluid, monoamine oxidase inhibitors, clorgyline, pargyline.
An analogous process of DBH release can be assumed to operate in the central nervous system, with cerebrospinal fluid (CSF) serving as a “sink” for released DBH and norepinephrine. Studies from this laboratory (Lerner, Goodwin, van Kammen, Post, Major, Ballenger and Lovenberg, 1978; Major, Lerner, Ballenger, Brown, Goodwin and Lovenberg, 1978) and others (Goldstein and Cubeddu, 1976; Okada, Ohta, Shinoda, Kato, Ikuta and Nagatsu, 1976; Fujita, Maruta, Teradaira, Beppu, Shinpo, Maeno, Ito, Nagatsu and Kato, 1977) show that DBH can be detected in human CSF, although at a concentration about l/5000 of that in serum. The source and the significance of DBH in CSF are unclear; CSF and serum levels of DBH are weakly, but significantly, correlated (Lerner et al., 1978; Fujita et a[., 1977). However, several lines of evidence suggest that this correlation does not result from DBH from plasma contaminating the CSF; it is more likely that CSF DBH levels are determined to some extent by the same genetic factors that determine serum DBH (Lerner et al., 1978). The changes in CSF DBH induced by changes in central noradrenergic activity and the relationship between DBH and norepinephrine in CSF have not been investigated previously. In this report, findings are presented on DBH and norepinephrine in CSF of depressed patients treated with monoamine oxidase inhibitors (MAOI’s). These drugs have been shown to decrease central noradrenergic activity in animals (D. W. Gallagher, personal communication). In the depressed patients, the MAOI’s caused a significant decrease in CSF DBH. In addition, DBH and norepinephrine were highly 423
N.P.18/4--G
PAULINELERNERet al.
424 correlated patients.
in CSF from drug-free
and MAOI-treated
EFFECT OF MONOAMINE OXIDASE INHIBITORS ON DBH ACTIVITY IN CSF
METHODS
1.6Subjects for this study were seven patients hospitalized for depression on a research ward of the National Institutes of Health in Bethesda, Maryland; CSF was obtained from all seven patients at the end of a three week period off all drugs. Patients were treated with clorgyline (20-30 mg/day) or pargyline (75100 mg/day) and were retested in the fourth week of treatment. Three of the patients in this group received both clorgyline and pargyline as part of a randomized, cross-over trial, with a three week placebo period between the trials with the two drugs. In each case, where clorgyline and pargyline data were available on the same patient, only one of these was used for the data analysis in this paper. Statistically equivalent results were obtained regardless of which of the two was used. Lumbar punctures (L.P.‘s) were performed at 9:00 a.m. following fasting and bed rest since midnight; L.P.‘s were nontraumatic, and CSF was free of blood (as determined by cell counts). Plasma was obtained during the same week, and usually on the same day, as the CSF. Norepinephrine in CSF from six patients was measured by a radioenzymatic method described elsewhere (Ziegler, Lake, Foppen, Shoulson and Kopin, 1976); CSF DBH was measured by a modification (Lerner et al., 1978) of the radioenzymatic method of Molinoff, Weinshilbourn, and Axelrod, (1971). Dopamine beta hydroxylase purified from bovine adrenal medullae (Wallace, Krantz and Lovenberg, 1973) was used to test recovery of the enzyme from CSF: in no case was significant enzyme inhibition observed. Dopamine beta hydroxylase in plasma was determined by the simpler, but less sensitive, spectrophotometric method (Nagatsu and Udenfriend, 1972). Although DBH values obtained by these two methods are not identical, they are strictly proportional to each other (Lovenberg, Bruckwick, Alexander, Horwitz and Keiser, 1974). RESULTS
g 1.4-is g 1.2E . l.Ok ; 0.8 y t >
0.6 -
4
0.4 -
g
0.2 ’
p ( 0.06
n=7
LDRUG- MONOAMINE FREE OXIDASE INHIBITORS
Fig. 1. Dopamine beta hydroxylase was measured in CSF from depressed patients receiving no medication (0), clorgyline (o), or pargyline (A). The data were analyzed by the two-tailed Student’s t-test for paired data. of these two compounds were highly correlated (r = 0.93, P < 0.01, n = 6). When data from drug-free and drug-treated patients were combined, there was, again, a strong correlation between norepinephrine and DBH in the CSF (r = 0.79, P < 0.005, n = 12; Fig. 2). Most of the points corresponding to MAOItreated patients in Figure 2 were above the regression line, while data points from drug-free patients were usually below the line. This suggests that the norepinephrine:DBH ratio was different in these two groups. This was investigated further by comparing the norepinephrine:DBH ratios for each patient off and on drug (Table 1). The MAO inhibitors caused a significant increased in this ratio (P < 0.05, n = 6).
Dopamine beta hydroxylase was measured in the CSF of each patient in the drug-free state and during treatment with monamine oxidase inhibitors. The DISCUSSION MAOI’s caused a signfiicant decrease (P < 0.05, n = 7) in CSF DBH (Fig. 1). The magnitude of the Clorgyline and pargyline have different specificities decrease ranged from approximately 5% to approxifor MAO type A and type B (Squires, 1972; Yang mately 50% and was not dependent on the initial and Neff, 1973). Clorgyline is a selective inhibitor of value. In contrast, these drugs had no significant effect MAO-A, which preferentially deaminates norepinephon plasma DBH. There were small differences (both rine and serotonin. Pargyline inhibits MAO-B, for increases and decreases) in plasma DBH; these which norepinephrine and serotonin are not preferred changes did not correlate with changes in the CSF substrates, but is much less selective than clorgyline enzyme. (Campbell, Robinson, Lovenberg and Murphy, 1978). When DBH and norepinephrine were measured in A measure of the specificity of these two drugs in CSF of unmedicated depressed patients, the amounts humans has been obtained by determination of
DBH and norepinephrine
RELATIONSHIP BETWEEN NOREPINEPHRINE AND DBH IN CSF
DBH ACTIVITY, nmole/ml/hr Fig. 2. Dopamine beta hydroxylase and norepinephrine were measured in CSF from unmedicated patients (0) and the same patients treated with monoamine oxidase inhibitors (A). 3-methoxy-4-hydroxyphenylethylglycol (MHPG), the major brain metabolite of norepinephrine, in CSF from patients in the current study. Both clorgyline and pargyline caused marked reductions in CSF MHPG, and the two drugs are equipotent in this respect at the doses used in this study (Major, Murphy, Lipper and Gordon, 1978). Because both drugs are potent inhibitors of the metabolism of norepinephrine by MAO, data obtained with these two drugs has been combined for the study of norepinephrine and DBH. The activity of DBH in CSF is partly determined by the rate of release of the enzyme into CSF. The present finding of a drug-induced decrease in CSF DBH is consistent with a decrease in the firing of central noradrenergic neurons in patients receiving MAO inhibitors for 3 to 4 weeks. These results are in agreement with the observation that the firing of noradrenergic neurons in the locus coeruleus in rats is sharply decreased after 3 weeks of treatment with MAO inhibitors (D. W. Gallagher, personal communication). Other possible explanations for the decrease in CSF DBH activity are enzyme inhibition or a decrease in tissue DBH. The present authors have
Table 1. Norepinephrine is in pg/ml and DBH in nmol/ml/hr. The monoamine oxidase inhibitors were clorgyline (a) and pargyline (b). The data were analyzed by the two-tailed Student’s t-test for paired data CSF NorepinephrineiCSF Drug-free Monoamine inhibitors
DBH
463
268
485
446
306
259
738b
275a
519b
557a
699b
879a
oxidase
Difference between p < 0.05, n=6.
ratios in drug-free
and drug-treated
patients:
in CSF
425
shown complete recovery of exogenous DBH added to the CSF samples, ruling out the presence of a reversible inhibitor of DBH. It is unlikely that the drugs inhibit DBH irreversibly since DBH does not use a flavin cofactor, and these drugs cause irreversible inhibition of MAO by binding covalently to the flavin-binding site on the enzyme. There are no reports on the effect of MAO inhibitors on DBH activity in brain tissue. This problem is now being investigated in studies on rat brain. The correlation between CSF DBH and plasma DBH and the very low activity of DBH in CSF as compared to plasma (Fujita et al., 1977; Lerner et al., 1978) raise the possibility that plasma is a significant source of DBH for the CSF. Evidence presented previously (Lerner et al., 1978), including negligible red cell counts in the CSF. indicates that the CSF samples are not contaminated with blood. However, the possibility of diffusion of minute amounts of DBH across the blood-CSF barrier could not be ruled out by direct experimental evidence. This question has been studied by measuring DBH in both plasma and CSF after treatment with MAO inhibitors and it has been shown that these drugs do not cause parallel changes in CSF DBH and plasma DBH. The changes in CSF DBH, rather than reflecting plasma enzyme changes, are probably more closely related to noradrenergic function in the central nervous system. The observed correlation between DBH and norepinephrine in CSF is consistent with the simultaneous release of these two compounds from noradrenergic storage vesicles during exocytosis. A similar phenomenon has been observed in vitro with guinea pig vas deferens, where there is a strong correlation between the amounts of DBH and norepinephrine released by depolarizing agents (Thoa, Wooten, Axelrod and Kopin, 1975). In human plasma, the levels of DBH and norepinephrine are not correlated (Lake et al., 1977), probably because of the large difference in the rates of clearance of the two compounds; the half-life of norepinephrine in plasma is several minutes, while the half-life of DBH is 8 to 12 hr (Kopin et al., 1976). In this study, a strong correlation has been observed between DBH and norepinephrine in CSF, and this finding has been confirmed with a larger group of drug-free depressed patients (unpublished data). When patients are treated with MAO inhibitors, the ratio of norepinephrine to DBH is increased. This suggests that MAO1 treatment causes an increase in vesicular norepinephrine, which is released with DBH during noradrenergic firing. Alternately, the MAO inhibitors may cause an increase in extravesicular norepinephrine, which could spill out from the neurons and be detected in CSF. The MAO inhibitors can affect CSF norepinephrine levels in two different ways. They can increase the amount of norepinephrine by inhibiting its breakdown, and they can decrease the amount of norepinephrine in CSF by depressing firing of noradrenergic neurons. These two opposing processes cause a net
PAULINELERNERet al.
426
increase in CSF norepinephrine in some patients and a net decrease in others; these changes are correlated with antidepressant response. These data are reported in detail elsewhere (Major, Lake, Murphy, Lipper, Lerner and Lovenberg, unpublished). In contrast, the consistent decrease in CSF DBH seen with MAO inhibitors is probably an indication of a decrease in noradrenergic activity in the patients. The activity of DBH in CSF is determined by both the tissue content of the enzyme and the rate of its release from neurons. The amount of DBH within the central nervous system is determined, at least in part, by genetic factors. The rate of release depends on noradrenergic firing rate and is subject to change by pharmacological and physiological factors. The decrease in CSF DBH caused by MAO inhibitors probably reflects a decrease in the firing of central noradrenergic neurons. The present results show that intraindividual changes in CSF DBH may be useful as a marker for changes in central noradrenergic activity in man.
REFERENCES Campbell, I. C., Robinson, D. S., Lovenberg, W. and Murphy, D. L. (1978). The effects of chronic regimens of clorgyline and pargyline on monoamine metabolism in the rat brain. J. Neurochem. Coyle, J. T. and Kuhar, M. J. (1974). Subcellular localization of dopamine+hydroxylase and endogenous norepinephrine in the rat hypothalamus. Brain Rex 65: 475-487. Fujita, K., Maruta, K., Teradaira, R., Beppu, K., Shinpo. K., Maeno, Y., Ito, T., Nagatsu, T. and Kato, T. (1977). Dopamine-b-hydroxylase activity in human cerebrospinal fluid and serum. J. Neurochem. 29: 1141-1142. Goldstein, D. J. and Cubeddu, L. X. (1976). Dopamine-/% hydroxylase activity in human cerebrospinal fluid. J. Neurochem. 26: 193-195. Hartman, B. K. (1974). Localization of the noradrenergic nervous system in human brain. J. Psychiat. Res. 11: 283-288. Kopin, I. J., Kaufman, S., Viveros, H., Jacobowitz, D., Lake, C. R., Ziegler, M., Lovenberg, W., and Goodwin, F. K. (1976). Dopamine-P-hydroxylase: basic and clinical studies. Ann. Int. Med. 85: 211-223. Lake, C. R., Ziegler, M. G. and Kopin, I. J. (1976). Use of plasma norepinephrine for evaluation of sympathetic neuronal function in man. Life Sci. 18: 1315-1326. Lake, C. R., Ziegler, M. G., Coleman, M. and Kopin, I. J. (1977). Lack of correlation of plasma norepinephrine and dopamine-fi-hydroxylase in hypertensive and normotensive subjects. Circ. Res. 41: 865-869. Lerner, P., Goodwin, F. K., van Kammen, D. P., Post,
R. M., Major, L. F., Ballenger, J. C. and Lovenberg, W. (1978). Dopamine-fl-hydroxylase in the cerebrospinal fluid of psychiatric patients. Biol. Psychiat. 13: 685-694. Lovenberg, W., Bruckwick, E. A., Alexander, R. W., Horwitz, D. and Keiser, H. R. (1974). Evaluation of serum dopamine-fi-hydroxylase activity as an index of sympathetic nervous activity in man. In: Neuropsychopharmacology of Monoamines and Their Regulatory Enzymes (Usdin, E., Ed.), pp. 129-134. Raven Press, New York. Major, L. F., Murphy, D. L. Lipper, S. and Gordon, E. (1978). Effects of clorgyline and pargyline on HVA, DOPAC, MHPG, VMA, and 5-HIAA levels in human cerebrospinal fluid. J. Neurochem. Major, L. F., Lerner, P., Ballenger, J. C., Brown, G. L., Goodwin, F. K. and Lovenberg, W. (1978). Dopamine beta hydroxylase in the cerebrospinal fluid: relationship to disulfiram-induced psychosis. Biol. Psychiat. Molinoff, P. B., Weinshilboum, R. and Axelrod, J. (1971). A sensitive enzymatic assay for dopamine-fi-hydroxylase. J. Pharm. exp. Ther. 178: 425-431. Nagatsu, T. and Udenfriend, S. (1972). Photometric assay of dopamine-fi-hydroxylase activity in human blood. Clin. Chem. 18: 980-984. Okada, T., Ohta, T., Shinoda, T., Kato, T., Ikuta, K. and Nagatsu, T. (1976). Dopamine-P-hydroxylase activity in serum and cerebrospinal fluid in neuropsychiatric diseases. Neuropsychobiology 2: 139-144. Slotkin, T. A. and Edwards, K. (1973). Effects of reserpine on the content and properties of rat adrenal medullary storage vesicles. Biochem. Pharmac. 22: 549-560. Smith, A. D., and Winkler, H. (1972). Fundamental mechanisms in the release of catecholamines. In: Catechofamines, Vol. 33 of Handbook of Experimental Pharmacology (Blaschko, H. and Muscholl, E., Eds), pp. 538-617. Springer, Berlin. Squires, R. F. (1972). Multiple forms of monoamine oxidase of intact mitochondria as characterized by selective inhibitors and thermal stability: A comparison of eight mammalian species. Adu. Biochem. Psychopharm. 5: 355-370. Thea, N. B., Wooten, G. F., Axelrod, J. and Kopin, I. J. (1975). On the mechanism of release of norepinephrine from sympathetic nerves induced by depolarizing agents and sympathomimetic drugs. Mol. Pharmac. 11: 10-18. Viveros, 0. H., Argueros, L. and Kirshner, N. (1968). Release of catecholamines and dopamine-b-oxidase from the adrenal medulla. Lqe Sci. 7: 609-618. Wallace, E. F., Krantz, M. L. and Lovenberg, W. (1973). Dopamine-fl-hydroxylase: a tetrameric glycoprotein. Proc. Natn. Acad. Sci., U.S.A. 70: 2253-2256. Weinshilboum, R. M. and Axelrod, J. (1971). Serum dopamine-b-hydroxylase. Decrease after chemical sympathectomy. Science 173: 931-934. Yang, H.-Y. T. and Neff, N. H. (1973). /j’-Phenylethylamine: a specific substrate for type B monoamine oxidase of brain. J. Pharm. exp. Ther. 187: 365-371. Ziegler, M. G., Lake; C. R., Foppen, F. J., Shoulson. I. and Kopin, I. J. (1976). Norepinephrine in cerebrospinal fluid. Brain Res. 108: 436440.
0028-3908/79/0401-0423X02.00/0
Vol. IS. pp. 423 lo 426
0 Pergamon Press Ltd 1979. Printed in Great Britain
DOPAMINE-#I-HYDROXYLASE AND NOREPINEPHRINE IN HUMAN CEREBROSPINAL FLUID: EFFECTS OF MONOAMINE OXIDASE INHIBITORS PAULINE LERNER’, L. F. MAJOR*, D. L. MURPHY’, S. LIPPER’, C. R. LAKES and W. LOVENBERG~ ‘Biological Psychiatry Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20014, U.S.A. ‘Clinical Neuropharmacology Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20014, U.S.A. jLaboratory of Clinical Science, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20014, U.S.A. and 4Hypertension-Endocrine Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20014, U.S.A. (Accepted 17 October 1978)
Summary-The effect of the monoamine oxidase inhibitors, clorgyline and pargyline, on central noradrenergic function in depressed patients was assessed by measuring dopamine beta hydroxylase (DBH) and norepinephrine in cerebrospinal fluid (CSF). These drugs caused a significant decrease in DBH in CSF. This decrease is consistent with a reduction in the release of DBH into CSF as noradrenergic firing is slowed by the drugs. Levels of DBH and norepinephrine in CSF were highly correlated. The ratio of CSF norepinephrine to CSF DBH increased when the patients were treated with monoamine oxidase inhibitors, consistent with a drug-induced increase in brain norepinephrine.
Dopamine-fl-hydroxylase (DBH), the enzyme which converts dopamine to norepinephrine, has been used as a marker for noradrenergic neurons in animals (Kopin, Kaufman, Viveros, Jacobowitz, Lake, Ziegler, Lovenberg and Goodwin, 1976) and humans (Hartman, 1974). It is localized along with norepinephrine in noradrenergic storage vesicles (Slotkin and Edwards, 1973; Coyle and Kuhar, 1974); when norepinephrine is released from these vesicles by exocytosis, the soluble fraction of DBH in the vesicles is also released (Viveros, Argueros and Kirshner, 1968; Smith and Winkler, 1972). Human serum contains a significant amount of DBH, which presumably originates from the sympathetic nervous system (Weinshilboum and Axelrod, 1971). The amount of DBH in human serum is largely determined by genetic factors, but can be changed, to a limited extent, by stressing the sympathetic nervous system (Kopin et al., 1976). The small magnitude of the observed changes in serum DBH levels may be explained by the large pool of DBH in serum and the slow turnover of the enzyme. Serum norepinephrine, like serum DBH, probably derives from sympathetic neurons. Marked changes in serum norepinephrine are seen when the sympathetic nervous system is stressed acutely (Lake, Ziegler, Coleman and Kopin, 1977). It has been suggested that serum norepinephrine is useful as a marker for sympathetic activity (Lake, Ziegler and Kopin, 1976). Key words : dopamine beta hydroxylase, norepinephrine, cerebrospinal fluid, monoamine oxidase inhibitors, clorgyline, pargyline.
An analogous process of DBH release can be assumed to operate in the central nervous system, with cerebrospinal fluid (CSF) serving as a “sink” for released DBH and norepinephrine. Studies from this laboratory (Lerner, Goodwin, van Kammen, Post, Major, Ballenger and Lovenberg, 1978; Major, Lerner, Ballenger, Brown, Goodwin and Lovenberg, 1978) and others (Goldstein and Cubeddu, 1976; Okada, Ohta, Shinoda, Kato, Ikuta and Nagatsu, 1976; Fujita, Maruta, Teradaira, Beppu, Shinpo, Maeno, Ito, Nagatsu and Kato, 1977) show that DBH can be detected in human CSF, although at a concentration about l/5000 of that in serum. The source and the significance of DBH in CSF are unclear; CSF and serum levels of DBH are weakly, but significantly, correlated (Lerner et al., 1978; Fujita et a[., 1977). However, several lines of evidence suggest that this correlation does not result from DBH from plasma contaminating the CSF; it is more likely that CSF DBH levels are determined to some extent by the same genetic factors that determine serum DBH (Lerner et al., 1978). The changes in CSF DBH induced by changes in central noradrenergic activity and the relationship between DBH and norepinephrine in CSF have not been investigated previously. In this report, findings are presented on DBH and norepinephrine in CSF of depressed patients treated with monoamine oxidase inhibitors (MAOI’s). These drugs have been shown to decrease central noradrenergic activity in animals (D. W. Gallagher, personal communication). In the depressed patients, the MAOI’s caused a significant decrease in CSF DBH. In addition, DBH and norepinephrine were highly 423
N.P.18/4--G
PAULINELERNERet al.
424 correlated patients.
in CSF from drug-free
and MAOI-treated
EFFECT OF MONOAMINE OXIDASE INHIBITORS ON DBH ACTIVITY IN CSF
METHODS
1.6Subjects for this study were seven patients hospitalized for depression on a research ward of the National Institutes of Health in Bethesda, Maryland; CSF was obtained from all seven patients at the end of a three week period off all drugs. Patients were treated with clorgyline (20-30 mg/day) or pargyline (75100 mg/day) and were retested in the fourth week of treatment. Three of the patients in this group received both clorgyline and pargyline as part of a randomized, cross-over trial, with a three week placebo period between the trials with the two drugs. In each case, where clorgyline and pargyline data were available on the same patient, only one of these was used for the data analysis in this paper. Statistically equivalent results were obtained regardless of which of the two was used. Lumbar punctures (L.P.‘s) were performed at 9:00 a.m. following fasting and bed rest since midnight; L.P.‘s were nontraumatic, and CSF was free of blood (as determined by cell counts). Plasma was obtained during the same week, and usually on the same day, as the CSF. Norepinephrine in CSF from six patients was measured by a radioenzymatic method described elsewhere (Ziegler, Lake, Foppen, Shoulson and Kopin, 1976); CSF DBH was measured by a modification (Lerner et al., 1978) of the radioenzymatic method of Molinoff, Weinshilbourn, and Axelrod, (1971). Dopamine beta hydroxylase purified from bovine adrenal medullae (Wallace, Krantz and Lovenberg, 1973) was used to test recovery of the enzyme from CSF: in no case was significant enzyme inhibition observed. Dopamine beta hydroxylase in plasma was determined by the simpler, but less sensitive, spectrophotometric method (Nagatsu and Udenfriend, 1972). Although DBH values obtained by these two methods are not identical, they are strictly proportional to each other (Lovenberg, Bruckwick, Alexander, Horwitz and Keiser, 1974). RESULTS
g 1.4-is g 1.2E . l.Ok ; 0.8 y t >
0.6 -
4
0.4 -
g
0.2 ’
p ( 0.06
n=7
LDRUG- MONOAMINE FREE OXIDASE INHIBITORS
Fig. 1. Dopamine beta hydroxylase was measured in CSF from depressed patients receiving no medication (0), clorgyline (o), or pargyline (A). The data were analyzed by the two-tailed Student’s t-test for paired data. of these two compounds were highly correlated (r = 0.93, P < 0.01, n = 6). When data from drug-free and drug-treated patients were combined, there was, again, a strong correlation between norepinephrine and DBH in the CSF (r = 0.79, P < 0.005, n = 12; Fig. 2). Most of the points corresponding to MAOItreated patients in Figure 2 were above the regression line, while data points from drug-free patients were usually below the line. This suggests that the norepinephrine:DBH ratio was different in these two groups. This was investigated further by comparing the norepinephrine:DBH ratios for each patient off and on drug (Table 1). The MAO inhibitors caused a significant increased in this ratio (P < 0.05, n = 6).
Dopamine beta hydroxylase was measured in the CSF of each patient in the drug-free state and during treatment with monamine oxidase inhibitors. The DISCUSSION MAOI’s caused a signfiicant decrease (P < 0.05, n = 7) in CSF DBH (Fig. 1). The magnitude of the Clorgyline and pargyline have different specificities decrease ranged from approximately 5% to approxifor MAO type A and type B (Squires, 1972; Yang mately 50% and was not dependent on the initial and Neff, 1973). Clorgyline is a selective inhibitor of value. In contrast, these drugs had no significant effect MAO-A, which preferentially deaminates norepinephon plasma DBH. There were small differences (both rine and serotonin. Pargyline inhibits MAO-B, for increases and decreases) in plasma DBH; these which norepinephrine and serotonin are not preferred changes did not correlate with changes in the CSF substrates, but is much less selective than clorgyline enzyme. (Campbell, Robinson, Lovenberg and Murphy, 1978). When DBH and norepinephrine were measured in A measure of the specificity of these two drugs in CSF of unmedicated depressed patients, the amounts humans has been obtained by determination of
DBH and norepinephrine
RELATIONSHIP BETWEEN NOREPINEPHRINE AND DBH IN CSF
DBH ACTIVITY, nmole/ml/hr Fig. 2. Dopamine beta hydroxylase and norepinephrine were measured in CSF from unmedicated patients (0) and the same patients treated with monoamine oxidase inhibitors (A). 3-methoxy-4-hydroxyphenylethylglycol (MHPG), the major brain metabolite of norepinephrine, in CSF from patients in the current study. Both clorgyline and pargyline caused marked reductions in CSF MHPG, and the two drugs are equipotent in this respect at the doses used in this study (Major, Murphy, Lipper and Gordon, 1978). Because both drugs are potent inhibitors of the metabolism of norepinephrine by MAO, data obtained with these two drugs has been combined for the study of norepinephrine and DBH. The activity of DBH in CSF is partly determined by the rate of release of the enzyme into CSF. The present finding of a drug-induced decrease in CSF DBH is consistent with a decrease in the firing of central noradrenergic neurons in patients receiving MAO inhibitors for 3 to 4 weeks. These results are in agreement with the observation that the firing of noradrenergic neurons in the locus coeruleus in rats is sharply decreased after 3 weeks of treatment with MAO inhibitors (D. W. Gallagher, personal communication). Other possible explanations for the decrease in CSF DBH activity are enzyme inhibition or a decrease in tissue DBH. The present authors have
Table 1. Norepinephrine is in pg/ml and DBH in nmol/ml/hr. The monoamine oxidase inhibitors were clorgyline (a) and pargyline (b). The data were analyzed by the two-tailed Student’s t-test for paired data CSF NorepinephrineiCSF Drug-free Monoamine inhibitors
DBH
463
268
485
446
306
259
738b
275a
519b
557a
699b
879a
oxidase
Difference between p < 0.05, n=6.
ratios in drug-free
and drug-treated
patients:
in CSF
425
shown complete recovery of exogenous DBH added to the CSF samples, ruling out the presence of a reversible inhibitor of DBH. It is unlikely that the drugs inhibit DBH irreversibly since DBH does not use a flavin cofactor, and these drugs cause irreversible inhibition of MAO by binding covalently to the flavin-binding site on the enzyme. There are no reports on the effect of MAO inhibitors on DBH activity in brain tissue. This problem is now being investigated in studies on rat brain. The correlation between CSF DBH and plasma DBH and the very low activity of DBH in CSF as compared to plasma (Fujita et al., 1977; Lerner et al., 1978) raise the possibility that plasma is a significant source of DBH for the CSF. Evidence presented previously (Lerner et al., 1978), including negligible red cell counts in the CSF. indicates that the CSF samples are not contaminated with blood. However, the possibility of diffusion of minute amounts of DBH across the blood-CSF barrier could not be ruled out by direct experimental evidence. This question has been studied by measuring DBH in both plasma and CSF after treatment with MAO inhibitors and it has been shown that these drugs do not cause parallel changes in CSF DBH and plasma DBH. The changes in CSF DBH, rather than reflecting plasma enzyme changes, are probably more closely related to noradrenergic function in the central nervous system. The observed correlation between DBH and norepinephrine in CSF is consistent with the simultaneous release of these two compounds from noradrenergic storage vesicles during exocytosis. A similar phenomenon has been observed in vitro with guinea pig vas deferens, where there is a strong correlation between the amounts of DBH and norepinephrine released by depolarizing agents (Thoa, Wooten, Axelrod and Kopin, 1975). In human plasma, the levels of DBH and norepinephrine are not correlated (Lake et al., 1977), probably because of the large difference in the rates of clearance of the two compounds; the half-life of norepinephrine in plasma is several minutes, while the half-life of DBH is 8 to 12 hr (Kopin et al., 1976). In this study, a strong correlation has been observed between DBH and norepinephrine in CSF, and this finding has been confirmed with a larger group of drug-free depressed patients (unpublished data). When patients are treated with MAO inhibitors, the ratio of norepinephrine to DBH is increased. This suggests that MAO1 treatment causes an increase in vesicular norepinephrine, which is released with DBH during noradrenergic firing. Alternately, the MAO inhibitors may cause an increase in extravesicular norepinephrine, which could spill out from the neurons and be detected in CSF. The MAO inhibitors can affect CSF norepinephrine levels in two different ways. They can increase the amount of norepinephrine by inhibiting its breakdown, and they can decrease the amount of norepinephrine in CSF by depressing firing of noradrenergic neurons. These two opposing processes cause a net
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increase in CSF norepinephrine in some patients and a net decrease in others; these changes are correlated with antidepressant response. These data are reported in detail elsewhere (Major, Lake, Murphy, Lipper, Lerner and Lovenberg, unpublished). In contrast, the consistent decrease in CSF DBH seen with MAO inhibitors is probably an indication of a decrease in noradrenergic activity in the patients. The activity of DBH in CSF is determined by both the tissue content of the enzyme and the rate of its release from neurons. The amount of DBH within the central nervous system is determined, at least in part, by genetic factors. The rate of release depends on noradrenergic firing rate and is subject to change by pharmacological and physiological factors. The decrease in CSF DBH caused by MAO inhibitors probably reflects a decrease in the firing of central noradrenergic neurons. The present results show that intraindividual changes in CSF DBH may be useful as a marker for changes in central noradrenergic activity in man.
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