- Email: [email protected]
Depression, antidepressants, and plasma DBH
P~ychiafry Research, 5, 33 l-334 ( 1981) ElsevieriNorth-Holland Biomedical Press
Depression, Roy J. Mathew,
331
Antidepressants,
and Plasma
Beng T. Ho, Chester
Davis, Dorothy
DBH Taylor, and Jon Reck
Received June 28, 1981; revised version received Ju1.v 21. 1981; accepted August
17. 1981.
Abstract. Plasma levels of dopamine-/3-hydroxylase (DBH) were determined in 16 unmedicated patients with major depressive episodes (nonpsychotic) and in an equal number of normal subjects, before and after 4 weeks of treatment with tricyclic antidepressants. Some eight patients were treated with amitriptyline, and the remainder received desipramine. The controls remained medication free during the entire experimental period. Degrees of depression were quantified before and after treatment with the Hamilton Rating Scale of Depression (HRSD). There were no significant differences between the depressed patients and the controls on levels of DBH. Similarly, there were no within-group, pre-posttreatment differences on the enzyme levels in either group. Pre- and posttreatment HRSD scores did not correlate with corresponding plasma DBH levels. Plasma levels of amitriptyline, nortriptyline (product of amitriptyline in the body), and desipramine at the end of 4 weeks of treatment also failed to correlate with the enzyme levels. Key Words. Dopamine-P-hydroxylase, desipramine.
depression,
amitriptyline,
nortriptyline,
(DBH) catalyzes the conversion of dopamine to norepinephrine. DBH is present in catecholamine-containing synaptic vesicles in sympathetic nerve terminals and adrenal medulla. Plasma DBH is derived from the enzyme expelled during the exocytotic release of epinephrine and norepinephrine following sympathetic stimulation (Cooper et al., 1978; Weinshilboum, 1979). Most investigators have found normal plasma levels of the enzyme in patients with depression (Shopsin et al., 1972; Wetterberg et al., 1972), but there are reports of elevated DBH levels in outpatients with depression (Friedman and Stolk, 1978) and decreases in activity of the enzyme in psychotic depressives (Meltzer et al., 1976). Little is known about the effect of antidepressants on plasma DBH; Friedman and Stolk (1978) reported no relationship between antidepressants and plasma levels of the enzyme, while a-methyl-dopa and minor tranquilizers were found to decrease and increase the levels of activity of the enzyme, respectively. Dopamine+hydroxylase
Methods Sixteen outpatients, 3 males and 13 females (mean age 37.1, SD 9.8) with a DSM-III(American Psychiatric Association, 1980) diagnosis of major depressive episode without psychotic symptoms participated in the project. History of a manic episode was not elicited from any subject.
Roy J. Mathew, M.D., is Chief, Psychosomatic Research Section, Texas Research Institute of Mental Sciences (TRIMS), 1300 Moursund, Houston, TX 77030, U.S.A. Beng T. Ho, Ph.D., is Chief, and Dorothy Taylor, M.S., is Biochemist, Neurochemistry and Neuropharmacology Research Section, TRIMS. Chester Davis, Ph.D., is Acting Chief, Instrumental Analysis Research Section, TRIMS. Jon Reck, Ph.D., is Chief, Outpatient
Clinic B, TRIMS.
0165-1781/81/0000-0000/$02.75
(Reprint
requests
to Dr. R.J. Mathew.)
@ Elsevier/North-Holland
Biomedical
Press
332 Most of the patients were medication free when they first entered the clinic; the others underwent a 2-week drug washout period. None of the subjects were on monoamine oxidase inhibitors or long-acting neuroleptics. Physical illnesses were excluded with history, physical examination, and routine laboratory tests. A control group of eight males and eight females were selected from physically and mentally healthy, drug-free volunteers. There were no significant age differences between the patients and their controls. DBH activity does not show gender differences (Weinshilboum, 1979). The study consisted of two evaluation sessions separated by 4 weeks of antidepressant treatment for the patient group and an identical drug-free interval for the controls. The Hamilton Rating Scale of Depression (HRSD) was used to quantify degrees of depression (Hamilton, 1960). Blood samples for DBH assay (10 ml) were drawn through a butterfly needle with heparin lock (to prevent blood from clotting in the needle) 10 minutes after the needle was inserted into a vein. The subjects relaxed in a quiet, semidark room, in supine position, during this interval. An additional 10 ml of blood were drawn during the posttreatment evaluation session to determine plasma levels of antidepressants. The subjects were instructed to avoid coffee, tea, and tobacco on the morning of the evaluation, and blood samples for enzyme assays were drawn at the same time of day, for both the index and control groups. While eight depressed patients received treatment with amitriptyline, the remaining ei~ght were treated with desipramine. The patients were instructed to avoid other medications and the controls to remain drug-free during this period. The patients were seen at weekly intervals, and the antidepressant dosage was adjusted based on their clinical response. Plasma drug level measurement was conducted only once during the posttreatment evaluation session. The blood sample for DBH assay was drawn in Korvac serum separation vacutainer tubes, allowed to clot for 30 minutes at room temperature, and then centrifuged at 1,200g for 10 minutes. Serum was then removed by a pipette and placed in a plastic storage tube and frozen immediately at -20°C. The activity of DBH was determined by a method based on the conversion of r4C-dopamine to t4C-norepinephrine followed by separation of the products on microcolumns of Dowex-50 in the ammonia (NH,+) form. The reaction mixture was that of Nagatsu et al. (1973). Serum samples were diluted I/ 10 and the copper sulfate concentration was 5 X lo-6M. DBH activity was expressed as nmoles of r4C-norepinephrine per ml per minute. The assay technique was validated by comparison with the values obtained via another assay procedure (Joh et al., 1974). Blood samples for plasma drug level determinations were drawn with a glass syringe and transferred into a glass tube. Tricyclic antidepressants were assayed by a procedure involving high-performance thin-layer chromatography (HPTLC) and in situ densitometric scanning (Fenimore et al., 1977, 1978; Fenimore and Meyer, 1979).
Results patients obtained a mean pretreatment HRSD score of 28.0 k SD 5.7; the posttreatment levels of depression were significantly lower than the pretreatment ones (posttreatment, mean HRSD 8.3 f. SD 6.6, t = 10.6, p < 0.001). Neither the depressed patients nor their controls showed any within-group differences on the DBH values obtained during the two evaluation sessions. Similarly, there were no significant intergroup differences on levels of activity of the enzyme at either evaluation session (Table 1). Pre- and posttreatment HRSD scores did not correlate with corresponding DBH values. Plasma levels for amitriptyline, nortriptyline (amitriptyline is converted into nortriptyline in the body), and desipramine at the posttreatment level were 68.3 I!I SD 21.6, 54.9 k SD 38.8, and 112.6 + SD 95.7, ng/ml, respectively. DBH values did not correlate with the tricyclic plasma levels.
The depressed
333 Table 1. Comparison and controls1
between pre- and posttreatment Pretreatment
Subjects
Mean
SD
DBH levels in patients
Posttreatment Mean
SD
t
P
Patients
(n = 161
2.09
0.64
2.31
0.73
1.06
0.30
Controls
cn = 16)
2.41
0.70
2.38
0.77
0.37
0.71
1. DBH values are expressed as nmoles of l”C-norepinephrine per ml per minute 2. Patients vs. controls at pretreatment level: t = 1.36, p < 0.18. 3. Patients vs. controls at posttreatment level: t = 0.26, p < 0.80.
Discussion The results reported here strongly argue against any association between plasma DBH and nonpsychotic depression. The study was conducted in a homogeneous group of depressed patients on whom the diagnosis of major depressive episode (nonpsychotic, unipolar) was carefully made. Nonspecific factors such as age, physical illnesses, pharmacological agents, and circadian rhythms were controlled. Since plasma DBH is derived from the enzyme present in the catecholamine synaptic vesicles in the presynaptic sympathetic neuron and released into the plasma along with catecholamines following sympathetic stimulation, conditions associated with release of catecholamines such as posture, environmental stimulation, exercise, and nicotine and coffee consumption are likely to increase plasma DBH and were therefore controlled for in this experiment (de Potter et al., 1969; Geffen et al., 1969; Weinshilboum et al., 1971; Levi, 1972). For example, the trauma associated with venipuncture has been demonstrated to produce an initial peak in plasma catecholamine levels. The use of a butterfly needle with a heparin lock and a blood sample withdrawn after a few minutes of venipuncture has been recommended to circumvent this source of error (Carruthers et al., 1970; Lake et al., 1976). However, it is questionable whether delaying withdrawal of the blood sample by 10 minutes would have remedied the above mentioned sources of error adequately, as DBH has been reported to have a half-life of more than 3 hours (Weinshilboum, 1979). The present findings do not contradict the report by Meltzer et al. (1976) of reduced levels of plasma DBH in depressed patients with psychosis, since the present group consisted of nonpsychotic, depressed patients. The report of increased DBH in depression by Friedman and Stolk (1978) was based on a heterogeneous sample of outpatients with physical, neurological, and psychiatric illnesses, medicated with a variety of drugs. The findings of that study are difficult to interpret because a diagnosis of depression was not established, and no attempts were made at subclassification. The present finding of absence of correlations between plasma levels of tricyclic antidepressants and DBH is in keeping with their findings. References American Psychiatric of Mental Disorders.
Association. 3rd ed. APA,
DSM-III: Washington,
Diagnostic DC (1980).
and
Statistical
Manual
334 Carruthers, M., Conway, N., Taggart, P., and Bates, D. Validity of plasma-catecholamine estimations. Lnncet, II, 62 (1970). Cooper, J.R., Bloom, F.E., and Roth, R.H. The Biochemical Basis of Neuropharmacology. Oxford University Press, New York (1978). de Potter, W.P., de Schaepdryver, A.F., Moerman, E.J., and Smith, A.D. Evidence for the release of vesicle proteins together with noradrenalin upon stimulation of the splenic nerve. Journal of Physiology, 204, 102 (1969). Fenimore, D.C., Davis, C.M., and Meyer, C.J. Determination of drugs in plasma by highperformance thin layer chromatography. Clinical Chemistr.v, 24 (8) 1386 (1978). Fenimore, D.C., and Meyer, C.J. A new approach to sample application in TLC. Journal of Chromatography,
186, 555 (1979).
Fenimore, D.C., Meyer, C.J., Davis, C.M., Hsu, F., and Zlatkis, J. High performance thinlayer chromatographic determination of psychopharmacological agents in blood serum. Journal of Chromatography, 142, 399 (1977). Friedman, M.J., and Stolk, J.M. Depression, hypertension and serum dopamine-betahydroxylase. Psychosomatic Medicine, 40, 107 (1978). Geffen, L.B., Livett, B.G., and Rush, R.A. Immunological localisation of chromogranins in sheep sympathetic neurones and their release by nerve impulses. Journal of Physiology, 204, 58 (1969).
Hamilton, chiatry,
M. A rating
scale for depression.
Journal
of Neurology,
Neurosurgery
and Psy-
23, 5 (1960).
Joh, T.H., Ross, R.A., and Reis, D.J. A simple and sensitive assay for dopamine-beta-hydroxylase. Anal.vtical Biochemistry, 62, 248 (1974). Lake, C.R., Ziegler, M., and Kopin, LJ. Use of plasma norepinephrine for evaluation of sympathetic neuronal function in man. Life Sciences, 18, 13 15 (1976). Levi, L. Stress and distress in response to psychosocial stimuli. Acta Medica Scandinavica, 191, Suppl. 528 (1972). Meltzer, H.Y., Cho, H.Y., Carroll, B.J., and Russo, P. Serum dopamine-beta-hydroxylase activity in the affective psychoses and schizophrenia. Archives of General Psychiatry, 33, 585 (1976).
Nagatsu, T., Thomas, P., Rush, R., and Udenfriend, S. A radio assay for dopamine-betahydroxylase activity in rat serum. Analytical Biochemistry, 55, 615 (1973). Shopsin, B., Freedman, L.S., Goldstein, M., and Gershon, S. Serum dopamine-beta-hydroxylase (DBH) activity and affective states. Psychopharmacologia. 27, 11 (1972). Weinshilboum, R.M. Serum dopamine-beta-hydroxylase. Pharmacological Reviews, 30 (2), 133 (1979).
Weinshilboum, R.M., Thoa, N.B., Johnson, N.B., and Axelrod, J. Proportional release of norepinephrine and dopamine-beta-hydroxylase from sympathetic nerves. Science, 174, 1349 (197 I). Wetterberg, L., Aberg, H., Ross, S.B., and Froden, 0. Plasma dopamine beta hydroxylase activity in hypertension and various neuropsychiatric disorders. Scandinavian Journal of Clinical and Laboratory Investigation, 30, 283 (1972).
Depression, Roy J. Mathew,
331
Antidepressants,
and Plasma
Beng T. Ho, Chester
Davis, Dorothy
DBH Taylor, and Jon Reck
Received June 28, 1981; revised version received Ju1.v 21. 1981; accepted August
17. 1981.
Abstract. Plasma levels of dopamine-/3-hydroxylase (DBH) were determined in 16 unmedicated patients with major depressive episodes (nonpsychotic) and in an equal number of normal subjects, before and after 4 weeks of treatment with tricyclic antidepressants. Some eight patients were treated with amitriptyline, and the remainder received desipramine. The controls remained medication free during the entire experimental period. Degrees of depression were quantified before and after treatment with the Hamilton Rating Scale of Depression (HRSD). There were no significant differences between the depressed patients and the controls on levels of DBH. Similarly, there were no within-group, pre-posttreatment differences on the enzyme levels in either group. Pre- and posttreatment HRSD scores did not correlate with corresponding plasma DBH levels. Plasma levels of amitriptyline, nortriptyline (product of amitriptyline in the body), and desipramine at the end of 4 weeks of treatment also failed to correlate with the enzyme levels. Key Words. Dopamine-P-hydroxylase, desipramine.
depression,
amitriptyline,
nortriptyline,
(DBH) catalyzes the conversion of dopamine to norepinephrine. DBH is present in catecholamine-containing synaptic vesicles in sympathetic nerve terminals and adrenal medulla. Plasma DBH is derived from the enzyme expelled during the exocytotic release of epinephrine and norepinephrine following sympathetic stimulation (Cooper et al., 1978; Weinshilboum, 1979). Most investigators have found normal plasma levels of the enzyme in patients with depression (Shopsin et al., 1972; Wetterberg et al., 1972), but there are reports of elevated DBH levels in outpatients with depression (Friedman and Stolk, 1978) and decreases in activity of the enzyme in psychotic depressives (Meltzer et al., 1976). Little is known about the effect of antidepressants on plasma DBH; Friedman and Stolk (1978) reported no relationship between antidepressants and plasma levels of the enzyme, while a-methyl-dopa and minor tranquilizers were found to decrease and increase the levels of activity of the enzyme, respectively. Dopamine+hydroxylase
Methods Sixteen outpatients, 3 males and 13 females (mean age 37.1, SD 9.8) with a DSM-III(American Psychiatric Association, 1980) diagnosis of major depressive episode without psychotic symptoms participated in the project. History of a manic episode was not elicited from any subject.
Roy J. Mathew, M.D., is Chief, Psychosomatic Research Section, Texas Research Institute of Mental Sciences (TRIMS), 1300 Moursund, Houston, TX 77030, U.S.A. Beng T. Ho, Ph.D., is Chief, and Dorothy Taylor, M.S., is Biochemist, Neurochemistry and Neuropharmacology Research Section, TRIMS. Chester Davis, Ph.D., is Acting Chief, Instrumental Analysis Research Section, TRIMS. Jon Reck, Ph.D., is Chief, Outpatient
Clinic B, TRIMS.
0165-1781/81/0000-0000/$02.75
(Reprint
requests
to Dr. R.J. Mathew.)
@ Elsevier/North-Holland
Biomedical
Press
332 Most of the patients were medication free when they first entered the clinic; the others underwent a 2-week drug washout period. None of the subjects were on monoamine oxidase inhibitors or long-acting neuroleptics. Physical illnesses were excluded with history, physical examination, and routine laboratory tests. A control group of eight males and eight females were selected from physically and mentally healthy, drug-free volunteers. There were no significant age differences between the patients and their controls. DBH activity does not show gender differences (Weinshilboum, 1979). The study consisted of two evaluation sessions separated by 4 weeks of antidepressant treatment for the patient group and an identical drug-free interval for the controls. The Hamilton Rating Scale of Depression (HRSD) was used to quantify degrees of depression (Hamilton, 1960). Blood samples for DBH assay (10 ml) were drawn through a butterfly needle with heparin lock (to prevent blood from clotting in the needle) 10 minutes after the needle was inserted into a vein. The subjects relaxed in a quiet, semidark room, in supine position, during this interval. An additional 10 ml of blood were drawn during the posttreatment evaluation session to determine plasma levels of antidepressants. The subjects were instructed to avoid coffee, tea, and tobacco on the morning of the evaluation, and blood samples for enzyme assays were drawn at the same time of day, for both the index and control groups. While eight depressed patients received treatment with amitriptyline, the remaining ei~ght were treated with desipramine. The patients were instructed to avoid other medications and the controls to remain drug-free during this period. The patients were seen at weekly intervals, and the antidepressant dosage was adjusted based on their clinical response. Plasma drug level measurement was conducted only once during the posttreatment evaluation session. The blood sample for DBH assay was drawn in Korvac serum separation vacutainer tubes, allowed to clot for 30 minutes at room temperature, and then centrifuged at 1,200g for 10 minutes. Serum was then removed by a pipette and placed in a plastic storage tube and frozen immediately at -20°C. The activity of DBH was determined by a method based on the conversion of r4C-dopamine to t4C-norepinephrine followed by separation of the products on microcolumns of Dowex-50 in the ammonia (NH,+) form. The reaction mixture was that of Nagatsu et al. (1973). Serum samples were diluted I/ 10 and the copper sulfate concentration was 5 X lo-6M. DBH activity was expressed as nmoles of r4C-norepinephrine per ml per minute. The assay technique was validated by comparison with the values obtained via another assay procedure (Joh et al., 1974). Blood samples for plasma drug level determinations were drawn with a glass syringe and transferred into a glass tube. Tricyclic antidepressants were assayed by a procedure involving high-performance thin-layer chromatography (HPTLC) and in situ densitometric scanning (Fenimore et al., 1977, 1978; Fenimore and Meyer, 1979).
Results patients obtained a mean pretreatment HRSD score of 28.0 k SD 5.7; the posttreatment levels of depression were significantly lower than the pretreatment ones (posttreatment, mean HRSD 8.3 f. SD 6.6, t = 10.6, p < 0.001). Neither the depressed patients nor their controls showed any within-group differences on the DBH values obtained during the two evaluation sessions. Similarly, there were no significant intergroup differences on levels of activity of the enzyme at either evaluation session (Table 1). Pre- and posttreatment HRSD scores did not correlate with corresponding DBH values. Plasma levels for amitriptyline, nortriptyline (amitriptyline is converted into nortriptyline in the body), and desipramine at the posttreatment level were 68.3 I!I SD 21.6, 54.9 k SD 38.8, and 112.6 + SD 95.7, ng/ml, respectively. DBH values did not correlate with the tricyclic plasma levels.
The depressed
333 Table 1. Comparison and controls1
between pre- and posttreatment Pretreatment
Subjects
Mean
SD
DBH levels in patients
Posttreatment Mean
SD
t
P
Patients
(n = 161
2.09
0.64
2.31
0.73
1.06
0.30
Controls
cn = 16)
2.41
0.70
2.38
0.77
0.37
0.71
1. DBH values are expressed as nmoles of l”C-norepinephrine per ml per minute 2. Patients vs. controls at pretreatment level: t = 1.36, p < 0.18. 3. Patients vs. controls at posttreatment level: t = 0.26, p < 0.80.
Discussion The results reported here strongly argue against any association between plasma DBH and nonpsychotic depression. The study was conducted in a homogeneous group of depressed patients on whom the diagnosis of major depressive episode (nonpsychotic, unipolar) was carefully made. Nonspecific factors such as age, physical illnesses, pharmacological agents, and circadian rhythms were controlled. Since plasma DBH is derived from the enzyme present in the catecholamine synaptic vesicles in the presynaptic sympathetic neuron and released into the plasma along with catecholamines following sympathetic stimulation, conditions associated with release of catecholamines such as posture, environmental stimulation, exercise, and nicotine and coffee consumption are likely to increase plasma DBH and were therefore controlled for in this experiment (de Potter et al., 1969; Geffen et al., 1969; Weinshilboum et al., 1971; Levi, 1972). For example, the trauma associated with venipuncture has been demonstrated to produce an initial peak in plasma catecholamine levels. The use of a butterfly needle with a heparin lock and a blood sample withdrawn after a few minutes of venipuncture has been recommended to circumvent this source of error (Carruthers et al., 1970; Lake et al., 1976). However, it is questionable whether delaying withdrawal of the blood sample by 10 minutes would have remedied the above mentioned sources of error adequately, as DBH has been reported to have a half-life of more than 3 hours (Weinshilboum, 1979). The present findings do not contradict the report by Meltzer et al. (1976) of reduced levels of plasma DBH in depressed patients with psychosis, since the present group consisted of nonpsychotic, depressed patients. The report of increased DBH in depression by Friedman and Stolk (1978) was based on a heterogeneous sample of outpatients with physical, neurological, and psychiatric illnesses, medicated with a variety of drugs. The findings of that study are difficult to interpret because a diagnosis of depression was not established, and no attempts were made at subclassification. The present finding of absence of correlations between plasma levels of tricyclic antidepressants and DBH is in keeping with their findings. References American Psychiatric of Mental Disorders.
Association. 3rd ed. APA,
DSM-III: Washington,
Diagnostic DC (1980).
and
Statistical
Manual
334 Carruthers, M., Conway, N., Taggart, P., and Bates, D. Validity of plasma-catecholamine estimations. Lnncet, II, 62 (1970). Cooper, J.R., Bloom, F.E., and Roth, R.H. The Biochemical Basis of Neuropharmacology. Oxford University Press, New York (1978). de Potter, W.P., de Schaepdryver, A.F., Moerman, E.J., and Smith, A.D. Evidence for the release of vesicle proteins together with noradrenalin upon stimulation of the splenic nerve. Journal of Physiology, 204, 102 (1969). Fenimore, D.C., Davis, C.M., and Meyer, C.J. Determination of drugs in plasma by highperformance thin layer chromatography. Clinical Chemistr.v, 24 (8) 1386 (1978). Fenimore, D.C., and Meyer, C.J. A new approach to sample application in TLC. Journal of Chromatography,
186, 555 (1979).
Fenimore, D.C., Meyer, C.J., Davis, C.M., Hsu, F., and Zlatkis, J. High performance thinlayer chromatographic determination of psychopharmacological agents in blood serum. Journal of Chromatography, 142, 399 (1977). Friedman, M.J., and Stolk, J.M. Depression, hypertension and serum dopamine-betahydroxylase. Psychosomatic Medicine, 40, 107 (1978). Geffen, L.B., Livett, B.G., and Rush, R.A. Immunological localisation of chromogranins in sheep sympathetic neurones and their release by nerve impulses. Journal of Physiology, 204, 58 (1969).
Hamilton, chiatry,
M. A rating
scale for depression.
Journal
of Neurology,
Neurosurgery
and Psy-
23, 5 (1960).
Joh, T.H., Ross, R.A., and Reis, D.J. A simple and sensitive assay for dopamine-beta-hydroxylase. Anal.vtical Biochemistry, 62, 248 (1974). Lake, C.R., Ziegler, M., and Kopin, LJ. Use of plasma norepinephrine for evaluation of sympathetic neuronal function in man. Life Sciences, 18, 13 15 (1976). Levi, L. Stress and distress in response to psychosocial stimuli. Acta Medica Scandinavica, 191, Suppl. 528 (1972). Meltzer, H.Y., Cho, H.Y., Carroll, B.J., and Russo, P. Serum dopamine-beta-hydroxylase activity in the affective psychoses and schizophrenia. Archives of General Psychiatry, 33, 585 (1976).
Nagatsu, T., Thomas, P., Rush, R., and Udenfriend, S. A radio assay for dopamine-betahydroxylase activity in rat serum. Analytical Biochemistry, 55, 615 (1973). Shopsin, B., Freedman, L.S., Goldstein, M., and Gershon, S. Serum dopamine-beta-hydroxylase (DBH) activity and affective states. Psychopharmacologia. 27, 11 (1972). Weinshilboum, R.M. Serum dopamine-beta-hydroxylase. Pharmacological Reviews, 30 (2), 133 (1979).
Weinshilboum, R.M., Thoa, N.B., Johnson, N.B., and Axelrod, J. Proportional release of norepinephrine and dopamine-beta-hydroxylase from sympathetic nerves. Science, 174, 1349 (197 I). Wetterberg, L., Aberg, H., Ross, S.B., and Froden, 0. Plasma dopamine beta hydroxylase activity in hypertension and various neuropsychiatric disorders. Scandinavian Journal of Clinical and Laboratory Investigation, 30, 283 (1972).