Psychiatry
35
Research, 32:3543
Elsevier
Generalized
Anxiety
Disorder:
Some
Biochemical
Dennis J. Munjack, Patricia L. Baltazar, Vincent DeQuattro, Ruby Palmer, Anicia Zulueta, Benjamin Cracker, Rudolf0 Galen Buckwalter, and Michele Leonard Received April 20, 1988; revised version received received September 5, 1989; accepted November
Aspects
Paul Sobin, Usigli,
February 2, 1989; second revised version 19, 1989.
Abstract. Fifty-one patients who met DSM-III criteria for generalized anxiety disorder, and who were recruited to participate in a drug outcome study, filled out a variety of rating scales and had blood samples drawn for plasma norepinephrine, epinephrine, and free 3-methoxy4hydroxyphenylglycol (M H PG) after a 20-min rest period. This group was compared to I5 normal controls who
also had their blood drawn after a 20-min rest period. While the two groups were initially found to have significantly different levels of plasma free M H PC through the use of t tests, this finding was not confirmed by subsequent discriminant analysis. Key Words. Generalized anxiety 3-methoxy-4-hydroxyphenylglycol.
disorder,
epinephrine,
norepinephrine,
Many of the somatic symptoms associated with anxiety can be produced by the sympathetic adrenal medullary discharge of catecholamines, the source of most peripheral epinephrine (Kelly, 1980). Increased peripheral adrenergic discharge has been reported in some but not all anxious patients (Chosy et al., 1970). In nonpatient populations, increased epinephrine (E) but not norepinephrine (NE) has been reported during public speaking (Dimsdale and Moss, 1980), during challenging mental tasks (Akerstedt et al., 1983), during dental procedures (Edmonson et al., 1972), in pilots landing on aircraft carriers (Rubin et al., 1970), and in students during exams (Jones et al., 1968). E has been shown in a number of studies to be more effective than NE in inducing both peripheral and central anxiety-like symptoms (Guttmacher et al., 1983). In patient populations, fi-adrenergic receptor antagonists such as propranolol
Dennis Munjack, M.D., is Associate Professor, Department of Psychiatry, California School of Medicine, and Director, U.S.C. Anxiety Disorders Clinic.
University of Southern
Patricia Baltazar, Ph.D., is Project Coordinator, U.S.C. Anxiety Disorders Clinic..Vincent DeQuattro, M.D., is Professor of Medicine, Universitv of Southern California School of Medicine. Paul Sobin. M.D., is Assistant Professor of Clinical Psychiatry, University of Southern California School of Medicine. Ruby Palmer, R.N., M.A., is Staff Nurse, U.S.C. Anxiety Disorders Clinic. Anicia Zulueta, M.D., is in private practice in Los Angeles, CA. Benjamin Cracker, M.D., is Assistant Director, U.S.C. Anxiety Disorders Clinic. Rudolf0 Usigli, M.D., is in private practice in Beverly Hills, CA. Galen Buckwalter, M.A., is Statistical Consultant, U.S.C. Anxiety Disorders Clinic. Michele Leonard is Research Assistant, U.S.C. Anxiety Disorders Clinic. (Reprint requests to Dr. D.J. Munjack, U.S.C. Anxiety Disorders Clinic, 1937 Hospital Place, L.A. County-U.S.C. Medical Center, Los Angeles, CA 90033, USA.) Ol65-1781/90/$03.50
@ 1990 Elsevier Scientific
Publishers Ireland
Ltd.
36 (Inderal) attenuate the somatic, and possibly the psychological, symptoms of anxiety (Pitts and Allen, 1979). In addition, patients with a hyperdynamic fi-adrenergic state, a condition not identical but clinically similar to an anxiety disorder, report an increased sensitivity to isoproterenol, a P-adrenergic agonist (Frohlich et al., 1969). These studies further point to peripheral adrenergic system involvement in human anxiety states. Studying patients with a DSM-III (American Psychiatric Association, 1980) diagnosis of agoraphobia with panic attacks, Ballenger (1984) found that the mean plasma NE of 36 agoraphobic patients was significantly higher than that of 12 normal controls. Nesse et al. (1984) reported in patients with panic attacks and mild agoraphobic symptoms that mean plasma E and NE levels were higher than in controls, although the differences were small. Similar results were reported by Cameron et al. (1984). Liebowitz et al. (1983) reported that elevated baseline E was the only variable that distinguished panic patients who went on to panic during lactate infusion from patients who did not panic after lactate infusion. However, there were no differences between patients and normal controls on any baseline catecholamine measures. These studies, based on measurement of peripheral sympathetic function, are consistent with the accepted physiology of anxiety (Uhde et al., 1984). The locus coeruleus and the central adrenergic system have also been implicated in anxiety states (Redmond, 1979; Redmond and Huang, 1979; Charney and Heninger, 1985a, 19856) prompting studies of the relationship of 3-methoxy-4-hydroxyphenylglycol (MHPG), the major metabolite of NE (Maas et al., 1976, 1979), to anxiety. MHPG has been used, in these studies, as an indirect measure of noradrenergic activity. Although MHPG originates largely from the periphery (Blombery et al., 1980), because of the high correlation between plasma levels of this metabolite and brain and cerebrospinal fluid (CSF) levels in laboratory animals (Elsworth et al., 1982) it is assumed that free MHPG diffuses easily across the blood-brain barrier (Kopin et al., 1983). Since central and peripheral noradrenergic pathways may be activated together (Crawley et al., 1978), plasma MHPG may provide a global index of overall noradrenergic activity. Levels of plasma MHPG have been significantly correlated with anxiety ratings in normals (Uhde et al., 1982; Ballenger et al., 1984) and phobic-anxious patients (Ko et al., 1983). Sheehan et al. (1984) reported a trend for MHPG excretion to be higher in patients with panic attacks than in controls. Garvey et al. (1978) found that patients with major depression and panic attacks had significantly higher 24-hour urinary excretion of MHPG than did patients with major depression and no panic attacks. Other studies implicating a dysregulation of noradrenergic function in anxiety disorder include Charney et al. (1983) and Sweeney et al. (1978). Charney et al. (1984) however, did not find elevated baseline plasma MHPG levels in panic attack patients, and they suggested that increased neuronal NE activity may only become apparent under certain conditions, such as stress. In one such “stress” condition, however, Woods et al. (1987) reported that exposure to phobic stimuli did not increase levels of plasma free MHPG in panic patients. Also, recent studies (Pohl et al., 1985; Uhde et al., 1988) have suggested no noradrenergic abnormality in anxiety states. Furthermore, no plasma MHPG rises have been observed during panic states
37
induced by lactate (Carr et al., 1986), carbon dioxide (Gorman et al., 1984), and caffeine (Charney et al., 1985). Patients with generalized anxiety disorder (GAD) have been reported to have plasma E and NE levels significantly higher than those of controls (Mathew et al., 1980). The same researchers, however, later failed to confirm elevations of plasma catecholamines in GAD patients, and suggested that their earlier findings might have occurred because blood samples were drawn immediately following venipuncture (Mathew et al., 1981). A review of the literature suggested that plasma MHPG comparisons between GAD patients and normals which control for the stressful effects of venipuncture are not presently available. We are reporting a comparison of GAD patients and controls using plasma measures of E, NE, and free MHPG drawn after a 20-min rest period. Methods Subjects. Subjects were part of a drug outcome study comparing propranolol (Inderal), chlordiazepoxide (Librium), and placebo in the treatment of GAD, and were recruited from advertisements placed in local newspapers and from referrals from physicians at the Los Angeles County-University of Southern California (L.A.C.-U.S.C.) Medical Center and from the surrounding communities. After the study was thoroughly explained, those subjects who met criteria were enrolled. The 26 female and 25 male GAD subjects enrolled were between the ages of 18 and 70; were in good health as determined by routine complete physical examinations, laboratory tests, and electrocardiograms; and met DSM-III criteria for the diagnosis of GAD as determined by a semistructured interview administered by an experienced clinician. Each GAD subject had a score of at least 20 on the Hamilton Rating Scale for Anxiety (Hamilton, 1969) and was rated to be at least moderately ill on the Clinical Global Impressions Scale (Department of Health, Education, and Welfare, 1976). All GAD subjects reported generalized, persistent anxiety manifested by symptoms from three of four symptom clusters (motor tension, autonomic hyperactivity, apprehensive expectation, and vigilance and scanning). In addition, the GAD subjects reported experiencing anxious mood continuously for at least I month and did not fall into any of the following exclusion categories: schizophrenia, major depressive disorder, organic brain syndrome, panic disorder, and alcohol or substance abuse. Also excluded were subjects who had any medical condition that would preclude the use of propranolol or chlordiazepoxide, or who had taken any psychoactive medication during the 2-week period before admission to the study. Some degree of social anxiety was acceptable, and did not constitute an exclusion criterion as long as the subject’s phobias or social fears did not dominate the clinical picture. Controls (I 2 females, 3 males) were volunteers recruited through verbal announcements from among the staff of the L.A.C.-U.S.C. Medical Center and did not meet DSM-III criteria for GAD or panic disorder as determined by semistructured clinical interview administered by the same clinician-investigators. In addition, none of the volunteers were currently in treatment with a mental health professional or taking psychoactive medications, and none of them were judged to be clinically depressed. Procedures. During the first clinic visit, the GAD subjects and drawn for the measurement of E, NE, and MHPG levels. The capped i.v. line that was kept patent with heparin and positioned the line was established, each GAD or control subject remained reduce the risk of catecholamine levels being influenced by the
controls had blood samples samples were drawn from a in an antecubital vein. After lying quietly for 20 min to nature of the venipuncture
38 procedure itself. The blood samples were taken at the end of this 20-min period. The E and NE measures were done by simultaneous single isotope radioenzymatic assays (Peuler and Johnson, 1977) while the MHPG measurement was done with high-performance liquid chromatography using electrochemical detection (Scheinin et al., 1983). In addition, the Hamilton Rating Scale for Anxiety (Hamilton, 1969), the Symptom Checklist-90 (SCL-90; Derogatis, 1977), and the Clinical Global impressions Scale (Department of Health, Education, and Welfare, 1976) were administered to all GAD subjects.
Results Fifty-one (5 I) subjects diagnosed as having GAD were compared with a group of 15 normal controls. Table 1 presents group means and standard deviations for age, E, NE, MHPG, and SCL-90 depression subscale scores. There was no significant difference between the groups with respect to age (t = 0.09, NS), but there was a significantly different sex distribution as indicated by a x2 procedure (x2 = 3.9, p < 0.05). With regard to catecholamine levels, there was no significant difference between the groups on NE (t = -0.42, p = 0.68) or E (l= 1.92, p = 0.06). However, levels of MHPG were significantly higher for GAD subjects than for controls (t = 2.09, p = 0.05). As shown in Table 1, the distribution of E and NE levels within the sample population appeared to be highly skewed, with large standard deviations. Scrutiny of frequency distribution data suggested that a few obviously outlying scores for E and NE might be responsible for the observed skewness. To identify outliers, a regression analysis with a plot of the standardized residuals was performed (Norusis, 1985). Any individuals with studentized residuals greater than the absolute value of 3 were identified as outliers and eliminated from subsequent analyses. When these scores were eliminated from the sample, the degree of skewness was reduced. Table 2 presents mean catecholamine levels after the elimination of outlying E and NE scores. There were still no significant differences between GAD and control subjects on either NE (t = 0.88, p = 0.39) or E (t = -1.48, p = 0.15).
Table 1. Mean (SD) age, E, NE, MHPG, and depression scores GAD Controls (n = 15)’
(n=51) Age
33.61
E (f-U)
77.86
(50.44)
56.07
(34.43)
377.63
(153.40)
400.20
(191.64)
17.42
(3.34) -
NE (ng/i) MHPG
(free,
pmol/ml)
Depression
(9.92)
19.79
(4.98)
19.46
(10.64)
33.40
-
(7.31)
Note. Depression scores are not available for controls, as they did not complete the Symptom Checklist-W E = epinephrine. NE = norepinephrine. MHPG = 3-methoxy-4-hydroxyphenylglycol. GAD = generalized anxiety disorder. 1. Except for MHPG, where n = 14.
Stepwise discriminant analysis was used to predict group membership (GAD vs. control) with age and sex entered in the equation first, followed by MHPG, NE, and E. This procedure was performed with all subjects for which complete data were
39
Table 2. Mean (SD) E, NE, and MHPG after elimination of outlying scores Control
GAD E (rig/U
71 .a2
(41.20)
56.07
(n = 49) NE (ng/l)
354.57
(101.81)
400.20
(191.64)
(n = 49) MHPG (pmol/ml)
19.79
(4.98) (n=51)
(34.43) (n = 15) (n = 15)
17.42
(3.34) (n = 14)
Note. E = epinephrine. NE = norepinephrine. MHPG = 3-methoxy-4-hydroxyphenylglycol. generalized anxiety disorder.
GAD =
available (GAD, n = 47; control, n = 14). None of the five variables significantly discriminated between GAD and control groups (see Table 3). To determine whether depression (as measured by the XL-90) might be related to observed catecholamine levels, correlation coefficients were obtained. No significant correlations were observed between depression and NE (r = -0.15, p = 0.31) E (r = -0.13,~ = 0.40), or MHPG (r = 0.1 I, p = 0.46). Finally, a one-way analysis of variance (ANOVA) was used to determine whether GAD subjects differed on catecholamine levels depending on which symptom type they rated as most prevalent: motor tension, autonomic hyperactivity, or apprehensive expectation. (Since few subjects endorsed vigilance and scanning as the most prevalent symptom type, they were eliminated from the analysis.) No statistically significant differences were found among the groups for E (F= 1.17; df = 1,62; p = 0.32) NE (F= 1.89; df = 1, 62;~ = 0.16) or MHPG (F= 1.15; df = 1, 63; p = 0.32).
Table 3. Discriminant analysis with age, sex, and catecholamine levels as predictors Variable
Wilks lambda
Significance level
Age
0.998
0.746
Sex
0.937
0.150
MHPG
0.901
0.111
NE E
0.872
0.098
0.830
0.062
Note. MHPG = 3-methoxy-4-hydroxyphenylglycol. NE = norepinephrine. E = epinephrine.
Discussion In animals, activation of the locus ceruleus is associated with fear behaviors and increased levels of NE and MHPG (the major metabolite of NE) in the brain, CSF, and plasma (Redmond, 1979; Redmond and Huang, 1979). These data point to the possibility that NE and MHPG might be markers for anxiety in humans. Plasma levels of NE reflect peripheral catecholaminergic activity (Uhde et al., 1984). There is accumulating evidence that the unconjugated or free form of MHPG is the major metabolite of NE in the human brain and provides a reliable estimate of
40 NE turnover in the brain (Maas et al., 1976; Karoum et al., 1977). However, the actual ‘% contribution of the brain to plasma MHPG is open to question. Estimates of the amount of peripheral MHPG that originates from the central nervous system have ranged from 10% to 60% (Maas et al., 1979). There is additional evidence that central and peripheral noradrenergic systems may be linked to each other and to the hypothalamic-pituitary-adrenal (HPA) axis (Sotsky et al., 1981; Elsworth et al., 1982; Jimerson et al., 1983). Previous studies of anxiety disorders, particularly panic disorder and agoraphobia with panic attacks, have partially supported the notion that noradrenergic activity is associated with alterations in state anxiety, but these findings have been variable and far from robust when peripheral plasma measures are used (Uhde et al., 1984). The results of the two articles reporting on plasma E and NE levels in GAD patients have been conflicting (Mathew et al., 1980, 1981). This is the third report of plasma NE and E levels in GAD patients and the first to report on MHPG. Our finding of no significant differences between GAD patients and controls on measures of E and NE is inconsistent with the evidence supporting noradrenergic system involvement in other anxiety disorders (Uhde et al., 1982; Ko et al., 1983; Ballenger et al., 1984). Although there was an initial finding of a significant difference between GAD and control groups on measures of plasma free MHPG using t tests, discriminant analyses to determine the strength of MHPG as a factor discriminating between GAD and control subjects yielded no significant results. The issue raised by Mathew et al. (198 1) about the stressful role of venipuncture was addressed in the present study. All patients and controls rested for 20 min with an indwelling capped i.v. line before blood samples were drawn for E, NE, and MHPG levels. It was assumed that the 20-min rest period was sufficient to eliminate the anxiety-inducing effect of venipuncture. Our results are consonant with this assumption and support findings by Mathew et al. (1981). We recommend, however, that in future studies, both patient and control groups complete anxiety rating scales contemporaneously with the drawing of blood. While caffeine intoxication was an exclusion criterion, our patients and controls were not required to restrict their caffeine intake during the 24 hours preceding the blood tests, as a prior study found that caffeine had no effect on MHPG (Uhde et al., 1984). There were no dietary restrictions imposed on subjects in the present study. Future studies evaluating the relationship between MHPG and anxiety need to address a number of variables that affect catecholaminergic function (e.g., motor activity, temperature, exercise, postural change, sexual arousal, mental tasks requiring concentration, and diet) (Levi, 1972; Dimsdale and Moss, 1980; Akerstedt et al., 1983; Kopin 1984). Since the degree of accompanying depression in anxious patients might explain the heterogeneity of catecholamine findings across patients with anxiety disorders (Garvey et al., 1987; Grunhaus, 1988), we measured depression levels using the SCL-90 in our patient sample. There was no correlation between depression levels and MHPG. This may well have resulted from our eliminating patients at screening who met criteria for major depressive disorder. It is possible, as suggested by Charney et al. (1984) that an obviously large catecholamine response might be apparent only under conditions of stress for
41 patients with generalized anxiety disorder. Yohimbine and clonidine provocation studies should be undertaken with generalized anxiety disorder patients. While DSM-III-R (American Psychiatric Association, 1987) distinguished between panic disorder, panic disorder with agoraphobia, and generalized anxiety disorder, it is not clear if these disorders differ with regard to catecholamine levels and MHPG levels. Elevations have been previously reported in panic disorder and panic disorder with agoraphobia (Nesse, 1984; Cameron et al., 1984). Our findings suggest that there might be biochemical differences port the DSM-III-R diagnostic distinction (Breier
between
the disorders
and sup-
et al., 1985). It is also possible, a more severe form of anxiety than does
however, that panic disorder represents GAD. Finally, a more accurate and complete picture of catecholamine function in GAD might be achieved by measuring catecholamine metabolites other than MHPG, as has been suggested recently in a study of catecholamine metabolism and disposition in depressed subjects (Maas et al., 1987).
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42
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35
Research, 32:3543
Elsevier
Generalized
Anxiety
Disorder:
Some
Biochemical
Dennis J. Munjack, Patricia L. Baltazar, Vincent DeQuattro, Ruby Palmer, Anicia Zulueta, Benjamin Cracker, Rudolf0 Galen Buckwalter, and Michele Leonard Received April 20, 1988; revised version received received September 5, 1989; accepted November
Aspects
Paul Sobin, Usigli,
February 2, 1989; second revised version 19, 1989.
Abstract. Fifty-one patients who met DSM-III criteria for generalized anxiety disorder, and who were recruited to participate in a drug outcome study, filled out a variety of rating scales and had blood samples drawn for plasma norepinephrine, epinephrine, and free 3-methoxy4hydroxyphenylglycol (M H PG) after a 20-min rest period. This group was compared to I5 normal controls who
also had their blood drawn after a 20-min rest period. While the two groups were initially found to have significantly different levels of plasma free M H PC through the use of t tests, this finding was not confirmed by subsequent discriminant analysis. Key Words. Generalized anxiety 3-methoxy-4-hydroxyphenylglycol.
disorder,
epinephrine,
norepinephrine,
Many of the somatic symptoms associated with anxiety can be produced by the sympathetic adrenal medullary discharge of catecholamines, the source of most peripheral epinephrine (Kelly, 1980). Increased peripheral adrenergic discharge has been reported in some but not all anxious patients (Chosy et al., 1970). In nonpatient populations, increased epinephrine (E) but not norepinephrine (NE) has been reported during public speaking (Dimsdale and Moss, 1980), during challenging mental tasks (Akerstedt et al., 1983), during dental procedures (Edmonson et al., 1972), in pilots landing on aircraft carriers (Rubin et al., 1970), and in students during exams (Jones et al., 1968). E has been shown in a number of studies to be more effective than NE in inducing both peripheral and central anxiety-like symptoms (Guttmacher et al., 1983). In patient populations, fi-adrenergic receptor antagonists such as propranolol
Dennis Munjack, M.D., is Associate Professor, Department of Psychiatry, California School of Medicine, and Director, U.S.C. Anxiety Disorders Clinic.
University of Southern
Patricia Baltazar, Ph.D., is Project Coordinator, U.S.C. Anxiety Disorders Clinic..Vincent DeQuattro, M.D., is Professor of Medicine, Universitv of Southern California School of Medicine. Paul Sobin. M.D., is Assistant Professor of Clinical Psychiatry, University of Southern California School of Medicine. Ruby Palmer, R.N., M.A., is Staff Nurse, U.S.C. Anxiety Disorders Clinic. Anicia Zulueta, M.D., is in private practice in Los Angeles, CA. Benjamin Cracker, M.D., is Assistant Director, U.S.C. Anxiety Disorders Clinic. Rudolf0 Usigli, M.D., is in private practice in Beverly Hills, CA. Galen Buckwalter, M.A., is Statistical Consultant, U.S.C. Anxiety Disorders Clinic. Michele Leonard is Research Assistant, U.S.C. Anxiety Disorders Clinic. (Reprint requests to Dr. D.J. Munjack, U.S.C. Anxiety Disorders Clinic, 1937 Hospital Place, L.A. County-U.S.C. Medical Center, Los Angeles, CA 90033, USA.) Ol65-1781/90/$03.50
@ 1990 Elsevier Scientific
Publishers Ireland
Ltd.
36 (Inderal) attenuate the somatic, and possibly the psychological, symptoms of anxiety (Pitts and Allen, 1979). In addition, patients with a hyperdynamic fi-adrenergic state, a condition not identical but clinically similar to an anxiety disorder, report an increased sensitivity to isoproterenol, a P-adrenergic agonist (Frohlich et al., 1969). These studies further point to peripheral adrenergic system involvement in human anxiety states. Studying patients with a DSM-III (American Psychiatric Association, 1980) diagnosis of agoraphobia with panic attacks, Ballenger (1984) found that the mean plasma NE of 36 agoraphobic patients was significantly higher than that of 12 normal controls. Nesse et al. (1984) reported in patients with panic attacks and mild agoraphobic symptoms that mean plasma E and NE levels were higher than in controls, although the differences were small. Similar results were reported by Cameron et al. (1984). Liebowitz et al. (1983) reported that elevated baseline E was the only variable that distinguished panic patients who went on to panic during lactate infusion from patients who did not panic after lactate infusion. However, there were no differences between patients and normal controls on any baseline catecholamine measures. These studies, based on measurement of peripheral sympathetic function, are consistent with the accepted physiology of anxiety (Uhde et al., 1984). The locus coeruleus and the central adrenergic system have also been implicated in anxiety states (Redmond, 1979; Redmond and Huang, 1979; Charney and Heninger, 1985a, 19856) prompting studies of the relationship of 3-methoxy-4-hydroxyphenylglycol (MHPG), the major metabolite of NE (Maas et al., 1976, 1979), to anxiety. MHPG has been used, in these studies, as an indirect measure of noradrenergic activity. Although MHPG originates largely from the periphery (Blombery et al., 1980), because of the high correlation between plasma levels of this metabolite and brain and cerebrospinal fluid (CSF) levels in laboratory animals (Elsworth et al., 1982) it is assumed that free MHPG diffuses easily across the blood-brain barrier (Kopin et al., 1983). Since central and peripheral noradrenergic pathways may be activated together (Crawley et al., 1978), plasma MHPG may provide a global index of overall noradrenergic activity. Levels of plasma MHPG have been significantly correlated with anxiety ratings in normals (Uhde et al., 1982; Ballenger et al., 1984) and phobic-anxious patients (Ko et al., 1983). Sheehan et al. (1984) reported a trend for MHPG excretion to be higher in patients with panic attacks than in controls. Garvey et al. (1978) found that patients with major depression and panic attacks had significantly higher 24-hour urinary excretion of MHPG than did patients with major depression and no panic attacks. Other studies implicating a dysregulation of noradrenergic function in anxiety disorder include Charney et al. (1983) and Sweeney et al. (1978). Charney et al. (1984) however, did not find elevated baseline plasma MHPG levels in panic attack patients, and they suggested that increased neuronal NE activity may only become apparent under certain conditions, such as stress. In one such “stress” condition, however, Woods et al. (1987) reported that exposure to phobic stimuli did not increase levels of plasma free MHPG in panic patients. Also, recent studies (Pohl et al., 1985; Uhde et al., 1988) have suggested no noradrenergic abnormality in anxiety states. Furthermore, no plasma MHPG rises have been observed during panic states
37
induced by lactate (Carr et al., 1986), carbon dioxide (Gorman et al., 1984), and caffeine (Charney et al., 1985). Patients with generalized anxiety disorder (GAD) have been reported to have plasma E and NE levels significantly higher than those of controls (Mathew et al., 1980). The same researchers, however, later failed to confirm elevations of plasma catecholamines in GAD patients, and suggested that their earlier findings might have occurred because blood samples were drawn immediately following venipuncture (Mathew et al., 1981). A review of the literature suggested that plasma MHPG comparisons between GAD patients and normals which control for the stressful effects of venipuncture are not presently available. We are reporting a comparison of GAD patients and controls using plasma measures of E, NE, and free MHPG drawn after a 20-min rest period. Methods Subjects. Subjects were part of a drug outcome study comparing propranolol (Inderal), chlordiazepoxide (Librium), and placebo in the treatment of GAD, and were recruited from advertisements placed in local newspapers and from referrals from physicians at the Los Angeles County-University of Southern California (L.A.C.-U.S.C.) Medical Center and from the surrounding communities. After the study was thoroughly explained, those subjects who met criteria were enrolled. The 26 female and 25 male GAD subjects enrolled were between the ages of 18 and 70; were in good health as determined by routine complete physical examinations, laboratory tests, and electrocardiograms; and met DSM-III criteria for the diagnosis of GAD as determined by a semistructured interview administered by an experienced clinician. Each GAD subject had a score of at least 20 on the Hamilton Rating Scale for Anxiety (Hamilton, 1969) and was rated to be at least moderately ill on the Clinical Global Impressions Scale (Department of Health, Education, and Welfare, 1976). All GAD subjects reported generalized, persistent anxiety manifested by symptoms from three of four symptom clusters (motor tension, autonomic hyperactivity, apprehensive expectation, and vigilance and scanning). In addition, the GAD subjects reported experiencing anxious mood continuously for at least I month and did not fall into any of the following exclusion categories: schizophrenia, major depressive disorder, organic brain syndrome, panic disorder, and alcohol or substance abuse. Also excluded were subjects who had any medical condition that would preclude the use of propranolol or chlordiazepoxide, or who had taken any psychoactive medication during the 2-week period before admission to the study. Some degree of social anxiety was acceptable, and did not constitute an exclusion criterion as long as the subject’s phobias or social fears did not dominate the clinical picture. Controls (I 2 females, 3 males) were volunteers recruited through verbal announcements from among the staff of the L.A.C.-U.S.C. Medical Center and did not meet DSM-III criteria for GAD or panic disorder as determined by semistructured clinical interview administered by the same clinician-investigators. In addition, none of the volunteers were currently in treatment with a mental health professional or taking psychoactive medications, and none of them were judged to be clinically depressed. Procedures. During the first clinic visit, the GAD subjects and drawn for the measurement of E, NE, and MHPG levels. The capped i.v. line that was kept patent with heparin and positioned the line was established, each GAD or control subject remained reduce the risk of catecholamine levels being influenced by the
controls had blood samples samples were drawn from a in an antecubital vein. After lying quietly for 20 min to nature of the venipuncture
38 procedure itself. The blood samples were taken at the end of this 20-min period. The E and NE measures were done by simultaneous single isotope radioenzymatic assays (Peuler and Johnson, 1977) while the MHPG measurement was done with high-performance liquid chromatography using electrochemical detection (Scheinin et al., 1983). In addition, the Hamilton Rating Scale for Anxiety (Hamilton, 1969), the Symptom Checklist-90 (SCL-90; Derogatis, 1977), and the Clinical Global impressions Scale (Department of Health, Education, and Welfare, 1976) were administered to all GAD subjects.
Results Fifty-one (5 I) subjects diagnosed as having GAD were compared with a group of 15 normal controls. Table 1 presents group means and standard deviations for age, E, NE, MHPG, and SCL-90 depression subscale scores. There was no significant difference between the groups with respect to age (t = 0.09, NS), but there was a significantly different sex distribution as indicated by a x2 procedure (x2 = 3.9, p < 0.05). With regard to catecholamine levels, there was no significant difference between the groups on NE (t = -0.42, p = 0.68) or E (l= 1.92, p = 0.06). However, levels of MHPG were significantly higher for GAD subjects than for controls (t = 2.09, p = 0.05). As shown in Table 1, the distribution of E and NE levels within the sample population appeared to be highly skewed, with large standard deviations. Scrutiny of frequency distribution data suggested that a few obviously outlying scores for E and NE might be responsible for the observed skewness. To identify outliers, a regression analysis with a plot of the standardized residuals was performed (Norusis, 1985). Any individuals with studentized residuals greater than the absolute value of 3 were identified as outliers and eliminated from subsequent analyses. When these scores were eliminated from the sample, the degree of skewness was reduced. Table 2 presents mean catecholamine levels after the elimination of outlying E and NE scores. There were still no significant differences between GAD and control subjects on either NE (t = 0.88, p = 0.39) or E (t = -1.48, p = 0.15).
Table 1. Mean (SD) age, E, NE, MHPG, and depression scores GAD Controls (n = 15)’
(n=51) Age
33.61
E (f-U)
77.86
(50.44)
56.07
(34.43)
377.63
(153.40)
400.20
(191.64)
17.42
(3.34) -
NE (ng/i) MHPG
(free,
pmol/ml)
Depression
(9.92)
19.79
(4.98)
19.46
(10.64)
33.40
-
(7.31)
Note. Depression scores are not available for controls, as they did not complete the Symptom Checklist-W E = epinephrine. NE = norepinephrine. MHPG = 3-methoxy-4-hydroxyphenylglycol. GAD = generalized anxiety disorder. 1. Except for MHPG, where n = 14.
Stepwise discriminant analysis was used to predict group membership (GAD vs. control) with age and sex entered in the equation first, followed by MHPG, NE, and E. This procedure was performed with all subjects for which complete data were
39
Table 2. Mean (SD) E, NE, and MHPG after elimination of outlying scores Control
GAD E (rig/U
71 .a2
(41.20)
56.07
(n = 49) NE (ng/l)
354.57
(101.81)
400.20
(191.64)
(n = 49) MHPG (pmol/ml)
19.79
(4.98) (n=51)
(34.43) (n = 15) (n = 15)
17.42
(3.34) (n = 14)
Note. E = epinephrine. NE = norepinephrine. MHPG = 3-methoxy-4-hydroxyphenylglycol. generalized anxiety disorder.
GAD =
available (GAD, n = 47; control, n = 14). None of the five variables significantly discriminated between GAD and control groups (see Table 3). To determine whether depression (as measured by the XL-90) might be related to observed catecholamine levels, correlation coefficients were obtained. No significant correlations were observed between depression and NE (r = -0.15, p = 0.31) E (r = -0.13,~ = 0.40), or MHPG (r = 0.1 I, p = 0.46). Finally, a one-way analysis of variance (ANOVA) was used to determine whether GAD subjects differed on catecholamine levels depending on which symptom type they rated as most prevalent: motor tension, autonomic hyperactivity, or apprehensive expectation. (Since few subjects endorsed vigilance and scanning as the most prevalent symptom type, they were eliminated from the analysis.) No statistically significant differences were found among the groups for E (F= 1.17; df = 1,62; p = 0.32) NE (F= 1.89; df = 1, 62;~ = 0.16) or MHPG (F= 1.15; df = 1, 63; p = 0.32).
Table 3. Discriminant analysis with age, sex, and catecholamine levels as predictors Variable
Wilks lambda
Significance level
Age
0.998
0.746
Sex
0.937
0.150
MHPG
0.901
0.111
NE E
0.872
0.098
0.830
0.062
Note. MHPG = 3-methoxy-4-hydroxyphenylglycol. NE = norepinephrine. E = epinephrine.
Discussion In animals, activation of the locus ceruleus is associated with fear behaviors and increased levels of NE and MHPG (the major metabolite of NE) in the brain, CSF, and plasma (Redmond, 1979; Redmond and Huang, 1979). These data point to the possibility that NE and MHPG might be markers for anxiety in humans. Plasma levels of NE reflect peripheral catecholaminergic activity (Uhde et al., 1984). There is accumulating evidence that the unconjugated or free form of MHPG is the major metabolite of NE in the human brain and provides a reliable estimate of
40 NE turnover in the brain (Maas et al., 1976; Karoum et al., 1977). However, the actual ‘% contribution of the brain to plasma MHPG is open to question. Estimates of the amount of peripheral MHPG that originates from the central nervous system have ranged from 10% to 60% (Maas et al., 1979). There is additional evidence that central and peripheral noradrenergic systems may be linked to each other and to the hypothalamic-pituitary-adrenal (HPA) axis (Sotsky et al., 1981; Elsworth et al., 1982; Jimerson et al., 1983). Previous studies of anxiety disorders, particularly panic disorder and agoraphobia with panic attacks, have partially supported the notion that noradrenergic activity is associated with alterations in state anxiety, but these findings have been variable and far from robust when peripheral plasma measures are used (Uhde et al., 1984). The results of the two articles reporting on plasma E and NE levels in GAD patients have been conflicting (Mathew et al., 1980, 1981). This is the third report of plasma NE and E levels in GAD patients and the first to report on MHPG. Our finding of no significant differences between GAD patients and controls on measures of E and NE is inconsistent with the evidence supporting noradrenergic system involvement in other anxiety disorders (Uhde et al., 1982; Ko et al., 1983; Ballenger et al., 1984). Although there was an initial finding of a significant difference between GAD and control groups on measures of plasma free MHPG using t tests, discriminant analyses to determine the strength of MHPG as a factor discriminating between GAD and control subjects yielded no significant results. The issue raised by Mathew et al. (198 1) about the stressful role of venipuncture was addressed in the present study. All patients and controls rested for 20 min with an indwelling capped i.v. line before blood samples were drawn for E, NE, and MHPG levels. It was assumed that the 20-min rest period was sufficient to eliminate the anxiety-inducing effect of venipuncture. Our results are consonant with this assumption and support findings by Mathew et al. (1981). We recommend, however, that in future studies, both patient and control groups complete anxiety rating scales contemporaneously with the drawing of blood. While caffeine intoxication was an exclusion criterion, our patients and controls were not required to restrict their caffeine intake during the 24 hours preceding the blood tests, as a prior study found that caffeine had no effect on MHPG (Uhde et al., 1984). There were no dietary restrictions imposed on subjects in the present study. Future studies evaluating the relationship between MHPG and anxiety need to address a number of variables that affect catecholaminergic function (e.g., motor activity, temperature, exercise, postural change, sexual arousal, mental tasks requiring concentration, and diet) (Levi, 1972; Dimsdale and Moss, 1980; Akerstedt et al., 1983; Kopin 1984). Since the degree of accompanying depression in anxious patients might explain the heterogeneity of catecholamine findings across patients with anxiety disorders (Garvey et al., 1987; Grunhaus, 1988), we measured depression levels using the SCL-90 in our patient sample. There was no correlation between depression levels and MHPG. This may well have resulted from our eliminating patients at screening who met criteria for major depressive disorder. It is possible, as suggested by Charney et al. (1984) that an obviously large catecholamine response might be apparent only under conditions of stress for
41 patients with generalized anxiety disorder. Yohimbine and clonidine provocation studies should be undertaken with generalized anxiety disorder patients. While DSM-III-R (American Psychiatric Association, 1987) distinguished between panic disorder, panic disorder with agoraphobia, and generalized anxiety disorder, it is not clear if these disorders differ with regard to catecholamine levels and MHPG levels. Elevations have been previously reported in panic disorder and panic disorder with agoraphobia (Nesse, 1984; Cameron et al., 1984). Our findings suggest that there might be biochemical differences port the DSM-III-R diagnostic distinction (Breier
between
the disorders
and sup-
et al., 1985). It is also possible, a more severe form of anxiety than does
however, that panic disorder represents GAD. Finally, a more accurate and complete picture of catecholamine function in GAD might be achieved by measuring catecholamine metabolites other than MHPG, as has been suggested recently in a study of catecholamine metabolism and disposition in depressed subjects (Maas et al., 1987).
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