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Adrenocorticotropin Hormone Response to Diazepam in Healthy Young Men Marc A. Schuckit, Richard Hauger, and Jeffery L. Klein
The effects of diazepam (DZ) infusions on changes in adrenocorticotropin hormone (ACTH) are a source of debate. In this study, 66 healthy young adult men were evaluated for changes in plasma ACTH after intravenous inJusions with placebo, 0.12 mg/kg and 0.20 mg/kg of DZ. After the higher DZ dose, 85% of the subjects demonstrated a decrease in ACTH of 20% or more, with the nadir occurring between 30 and 60 min postinfusion and values returning to baseline levels by 180 rain. These results support the conclusion that at the clinically relevant doses used here, DZ infusions are associated with a significant decrease in ACTH. Introduction Studies of the hypothalamic-pituitary-adrenal (HPA) axis have a unique place in medicine and psychiatry. Aberrations in plasma levels and circadian patterns of corticotropin releasing factor (CRF), adrenocorticotropin hormone (ACTH), and cortisol are important in the diagnosis of a number of tumors, they have been studied as potential causes or mitigating factors in major depression, alcoholism, and sleep disorders, and have helped further our understanding of physiological responses to stress (File 1990; De Souza 1990). Our knowledge of the clinical implications and neurochemical controls of the HPA axis has been advanced by the development of systematic perturbations of the system. These include observations of the effects of stress, infusions of hormones such as insulin, and challenges with various medications. The optimal interpretation of results of such studies requires carefully described methods with large enough samples to generate reliable data, because it is unlikely that any single type of challenge will result in a 100% subject response. Reflecting the normal circadian fluctuations, it is also important to control for placebo effects, and for human studies, the challenges must also be safe. F~'lr the latter reason, evaluations of neurohormonai responses to benzodiazepines are attractive. Following such drugs, alterations in cortisol and ACTH are reported to be dose dependent, with increases likely to be observed after very high dose infusions in animals (e.g., 10 mg/kg of DZ), and some indication of decreases in hormone plasma levels after lower doses (e.g., 0.1-1.0 mg/kg) (Lakic et al 1986; File 1990; De Souza 1990; Kalogeras et al 1990). Most studies agree that these lower, more physiological
From the University of California-San Diego. School of Medicine (MAS, RH, JLK) and Alcohol Research Center, Veterans Affairs Medical Center (MAS), San Diego, CA. Received March 5. 1991, revised November 15, 1991. Address reprint requests to Marc A. Schuckit, M.D., Veterans Affairs Medical Center (I J6A), 3350 La Jolla Village Drive, San Diego, CA 92161. © 1992 Society of Biological Psychiatry
0006-3223/92/$05.00
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levels of benzodiazepines attenuate stress-induced or insulin-induced increases in both cortisol and ACTH (Roy-Byrne et al 1988; De Souza 1990; File 1990), and diminish hormone levels in patients with physiological conditions that result in hypersecretion of the HPA axis (Gram and Christensen 1986; De Souza 1990). There is general, but not unanimous, agreement that in the absence of these conditions, physiological doses of a variety of benzodiazepines result in a decrease in cortisol (Hommer et al 1986; Laakmann 1984; Schuckit et al 1991a), but there is more discord regarding changes in ACTH. Some studies indicate a decrease in this hormone similar to that observed with cortisol (Risby et al 1989; Owens et al 1989), but others relate possible increases or no change, or are difficult to interpret data (Tormey and Darraugh 1979; Roy-Byn~e et al 1988; Calogero et al 1990). It is possible that the disparities regarding ACTH might reflect small samples and the variance added by including patients with medical, psychiatric, and substance use problems. Results from samples of 10 or fewer subjects al~o make it difficult to estimate the proportion of individuals who reliably demonstrate a decrease in ACTH following benzodiazepines. In addition, the sensitivity of the usual double-antibody radioimmunoassays for measuring ACTH may not be sufficient to detect inhibition of basal circulating levels. The developmentof a highly sensitive immunoradiometric assay for ACTH now provides a more sensitive method of measuring decreases in circulating ACTH levels below the baseline. The present article presents data on a sample of 6o ,:arefully screened young men for whom ACTH values are available following slow intravenous (iv) infusions of placebo and two clinically relevant doses of DZ.
Methods The 66 men, including 36 who were incorporated in other relevant articles (Schuckit et al 1991a, 1991b; Monteiro et ai 1990) were originally identified between 1985 and 1988 through a structured questionnaire mailed to male students and nonacademic staff at the University of California, San Diego. The subjects were screened further by a subsequent telephone interview with themselves and a resource person when necessary, and with a semistructured personal face-to-face interview. These instruments were used to exclude potential subjects who were alcohol abstainers, whoa met criteria for DSM-III Axis 1 disorders including those related to substance use, or who had medical problems or were taking medications likely to interfere with a drug challenge. As part of a larger investigation, the sample includes subjects who have a biological father who met DSM-III criteria for alcohol abuse or dependence [family history-positive (FHP) men] as well as family history-negative (FHN) controls (Schuckit et al 1991a). After initial selection, subjects were brought to the laboratory for a total of four 9 AM infusions, each separated by a minimum of 72 hr. The men and the laboratory personnel were both blind to the drug condition. The information presented here focuses on data gathered during sessions when subjects were challenged, in random order, with placebo, 0.12 and 0.20 mg/kg of DZ infused over 7 min. The fourth session involved an oral alcohol challenge, generating data not relevant to the hormonal responses to benzodiazepines. The DZ doses were chosen to create intensities of subjective intoxication that are similar to those observed following 0.75 and 1. I ml/kg of ethanol, the doses used historically in our laboratory since the mid-1970s (Schuckit et al 1991a, 1991b). Subjects fasted overnight, arriving in the laboratory at 7:00 AM at which time they were given a light breakfast. Two heparin-locks were inserted into separate antecubital veins in the
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nondominant ann, with one used for the infusion and the other for withdrawal of blood samples. After a minimum of 30 min for acclimation, the infusions were carried out at baseline, and blood samples were withdrawn every half-hour over the 180 rain for analyses of DZ desmethyldiazepam (DMZ), and ACTH plasma levels. During the test sessions, subjects also participated in a series of evaluations of subjective feelings, motor performance, and cognition and, thus, no individual was allowed to fall asleep. Because the protocol included a wide range of tests, time constraints required that blood samples could not be taken more frequently. Blood for ACTH was collected in EDTA vacutainer tubes on ice and separated into plasma by centrifugation at 1000 x g with 90 min of collection. Plasma ACTH concentrations were measured with a highly sensitive and specific immunoradiometric assay that utilizes two antibodies, both with high affinity and specificity for defined regions of the ACTH molecule (Raft and Findling 1989). Following these methods, the highest concentration of ACTH measurable without dilution is approximately 1700 pg/ml, and the limit of detection (assay sensitivity) is 1.0 pg/ml, lntraassay and interassay coefficients of variation are approximately 3% and 7%, respectively. Benzodiazepine plasma levels were determined by an electron-capture gas-liquid chromatography procedure (Greenblatt et al 1980). The major data analytic procedures evaluated ACTH levels following the infusions utilizing a 3 (dose: placebo, low, high) x 6 (hormone sampling time points) repeated measures analysis of variance (ANOVA). The assessment of change during the placebo session was carried out with a single six time point ANOVA, and a subsequent determination of potential differenceg between the reactions to the two different DZ doses was gauged with a multiple points analysis of covariance (ANCOVA), covarying for placebo in a 2 (dose: low, high) x 6 (hormone sampling time points) procedure. Similar analyses were carried out for cortisol. The comparability of the three baseline ACTH levels was evaluated with an ANOVA, revealing no significant difference (F = 0.06; 2, 130 df; p = 0.94); nor were there baseline differences for the subset of men for cortisol (F -0.58; 2, 70 df; p = 0.56). The 66 men were all Caucasian (Schuckit et al 1991a; Schuckit 1990). The average individual was 22. I .4- 1.98 years of age, had completed 15.4 +- I. 18 years of school, weighed 165.1 -+ 22.97 pounds, and consumed alcoholic beverages on 7.6 _ 4.66 days per month, taking 3.2 -+ 1.58 drinks per occasion in the 6 months prior to testing. A drink was defined as 4 ounces of wine, 12 ounces of beer, or 1.5 ounces of 80-proof beverage. No subject had ever used benzodiazepines for a "high." Results During the active challenges, the DZ and DMZ plasma levels peaked by the time of the first blood samples at 30 min, with the diazepam plasma levels returning slowly toward baseline, and the DMZ levels plateauing throughout the experiment, as shown in Table 1. Because of procedural problems, these results were available on only 55 of the 66 men (83%), but are similar to those described in prior publications (Monteiro et al 1990; Schuckit et al 1991a). Figure 1 presents the ACTH values ( -- SEM) at baseline and each half-hour over the 180 min in the three test conditions. Consistent with the time of day during which the samples were gathered, the values during placebo session slowly decreased over time, with an ANOVA revealing a significant time effect (F = 3.83; 5,325 dr; p -- 0.002).
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Table 1. Mean ( "4-SD) for Blood Levels of Diazepam (ng/ml) and Desmethyldiazepam (ng/ml) Following the Active Infusions Dose :
O.12 mg/kg
Time (min)
DZ
30 60 90 120 150 !80
373.5(108.26) 283.5(69.98) 218,6(55,06) 184,0(49,68) 151,5(36.66) 134,2(33.53)
0.20 mg/kg DMZ 15.2(14.92) 16.7(!4 . 4 2 ) i 7,6(14.36) 18.1(14.11) 17,5(13.39) 18,3(!3 . 8 9 )
DZ
DMZ
635.5(124.37) 456.6(98.00) 353. I(90.64) 301.1(74.29) 25I. 3(57.69) 221.7(55.67)
18.6(! I. 32) 22.2(! 1.35) 23.5(12.32) 24.1(11.71) 24.3(! 1.58) 25.2( I 1.55)
In contrast to these results, following both the low-dose and high-dose DZ infusions, there was a rapid and marked decrease in ACTH values by 30 rain, a plateau until 60 min, and then a slow return toward baselim: over the remainder of the experiment. The repeated measures ANOVA considering all three doses and six postbaseline time points revealed a significant dose effect (F = 44.28; 2,130 df; p < 0.0001), and a trend for a main time effect (F ffi 2.07; 5,325 df; p ffi 0.07). There was a significant dose by time interaction (F = 9.04; 10,650; p < 0.0001). Potential differences in ACTH values following low-dose versus high-dose DZ as measured by the ANCOVA covarying for placebo results revealed a trend for a significant dose main effect (F ffi 3.63; 1,65 df; p = 0.06), a significant time main effect (F ffi 15.13; 5,324 df; p < 0.0001), and a nonsignificant dose by time interaction (F = 0.45; 5,325 df; p ffi 0.82). Data on the response of cortisol during similar DZ infusions have been presented elsewhere (Schuckit et al 1991a). Due to budgetary constraints, only 36 of the 66 men
40
3O
E m w L
Figure 1. Plaslaa ACTH response (mean - SEM) to IV infusions of placebo, 0.12 mg/kg DZ, and 0.20 mg/kg DZ over time in 66 healthy men. 10
0
D Plaoebo V Low Dose Olozepam • High Dose Dlozeporn . , , l s i 30 60 90 120 150 Po#t-lnfusTon Time (mlnutee)
! t80
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Table 2. Pearson Correlations of Blood Chemistry Measures Over 180 Min 0.12 mg/kg DZ infusion Correlations for
30
60
90
120
150
180
Summary
DZ with ACTH DZ with Cortisoi DMZ with ACTH DMZ with Cortisol ACTH with Cortisol
-0.11 -0.45 0. I ! -0.09 0.32
-0.11 -0.35 0.08 -0.04 0.06
-0.14 -0.15 0.03 -0.91 0.19
-0.18 -0.15 0.03 0.05 0.22
-0.22 -0.32 0.02 0.00 0.34
-0.13 -0.27 0.03 0.17 0.22
-0.2.~, -0.27 0.00 -0.13 0.41
-0.11 -0.33 0.00 -0.33 0.23
-0.34 -0.23 -0.19 -0.36 0,48
0.20 mg/kg DZ infusion DZ with ACTH DZ with Cortisol DMZ with ACTH DMZ with Cortisol ACTH with Cortisol
-0.28 -0.23 -0.04 -0,07 0.17
-0.20 -0.23 -0.04 -0.07 0.24
-0.05 -0.24 -0.04 -0.16 0.48
-0.12 -0.19 -0.10 -0.21 0.54
-0.11 -0.28 -0.05 -0.33 0.46
reported here also had cortisoi values available, and these have been analyzed in preparation for the evaluation of the relationships of hormone levels to drug plasma levels presented in Table 2. As shown in Figure 2, the decreases in cortisol over time related here are quite similar to our prior report, which had included these 36 men. Regarding cortisol levels following placebo, the ANOVA revealed a significant time effect (F = 13.72; 5,175 df; p < 0.001). For the three doses, the ANOVA revealed significant dose ( F = 17.96; 2,70 df; p < 0.001) and time (F = 24.60; 5,175 df; p < 0.001) main effects, but a nonsignificant dose-by-time interaction ( F = 1.46; 10,350 df; p = 0.16). After covarying for placebo effects in an ANCOVA, the dose main effect was nonsignificant (F = 2.99; 1,35 df; p = 0.09), time was significant ( F = 10.46; 5,174 df;
20
10 16
~ 14
o..
to i
g
R
6 4 2 0
Figure 2. Plasma cortisol response (mean ± SEM) to IV infusions of placebo, 0.12 mg/kg DZ, and 0.20 mg/kg DZ over time in 36 healthy men, a subset of the 65 men in Figure !.
a Placebo V Low Don Dlozepom • High Dose Dlazepom !
DA
30 60 90 t20 150 Poat-lnfuelon Time (mlnutce)
180
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Table 3. Proportion of Subjects with ACTH Decreases of 20% or More from Baseline Following Three Challenges Time Challenge Placebo 0.12 mg/kg DZ 0.20 mg/kg DZ
30 Min (%)
60 Min (%)
27 6! 85
33 79 79
p < 0.001), and the dose-by-time interaction was not significant (F = 0.55; 5,175 df, p = 0.74). These indicate a lack of a significant difference for the two DZ doses on cortisol. The data on cortisol are mentioned here so that the relationship among cortisoi, ACTH, DZ. and DMZ blood levels can be presented on these men in Table 2. The correlations are presented as they relate to the full set of data: 55 men had ACTH and DZ levels available but only 26 had cortisol and DZ. The values are similar if only those 26 men on whom all values were available were used in the analyses. Of course, after both low and high dose challenges, ACTH and cortisol values are positively correlated. Also DZ plasma levels evidence a moderate negative correlation with both ACTH and cortisol across both experimental conditions, with little evidence of differences in the magnitude of these correlations for either the dose or the hormone. DMZ, on the other hand, showed little correlation with ACTH, but a more intense correlation with cortisol, especially after the high dose infusions. A significant change in ACTH following DZ for a sample of 66 men could occur even if a relatively large subgroup demonstrated no alteration in the hormone after the infusion. Unfortunately, the literature is of little help identifying what magnitude of change is clinically relevant. Thus, arbitrarily we evaluated the proportion of men who demonstrated a decrease of between 10% and 30% from baseline values, finding the most prominent differences between placebo and high-dose DZ at a 20% decrement at 30 min. Table 3 demonstrates that although such a change is seen at 30 min in only 27% of the men after placebo, it was observed in 85% after high-dose DZ. An effort was made to determine if the men with such decreases in ACTH after DZ differed from the remainder on relevant va, i hies, but the two groups proved to be similar on all tested characteristics including ago, weight, family history of alcoholism, and their drinking patterns over the prior 6 months. The larger study fi'om which these data were extracted was originally created to determine if sons of alcoholic fathers, themselves at fourfold increased risk for future alcoholism (Schucki~ 1990), differed from well-matched controls on their responses to alcohol and DZ challenges, Though earlier studies have consistently revealed a less intense response for the FHP men following alcohol (Schuckit 1990), there were no indications of a similar phenomenon after infusions of DZ (Schuckit et al 1991a, 1991b). Similar to those earlier results, when the 66 men reported on here were divided into the component 33 FHPs and 33 FHNs, no significant differences nor consistent trends differentiating the family groups were noted for ACTH following any of the three infusion,~. For example, a 3 (dose: placebo, low, high) × 2 (FHP,FHN) x 6 (hormone s~:mpling time points) ANOVA revealed no significant family history main effect (F = 0.99; 1,64 df; p = 0.32), a nonsignificant family history by dose interaction (F = 1.64; 2,128 dr; p =
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0.20), a nonsignificant family history by time factor (F = 1.43; 5,320 df; p -- 0.21), and a nonsignificant family history by dose by time interaction (F = 0.81; 10,640 df; p ffi 0.62). Discussion This study demonstrated that IV infusions of clinically relevant levels of DZ resulted in a rapid decrease in plasma ACTH levels that reversed within 3 hr. These findings were generated in a large sample of healthy young men for whom psychiatric and substance use disorders, medical problems, and recent ingestion of medications were controlled. Even after considering the observation that approximately one-quarter of the subjects demonstrated a 20% or greater decrease in this hormone following placebo, a similar diminution in hormone levels was observed in 85% of the subjects following an active DZ infusion. The reasons for the variability between subjects for the ACTH response to DZ are not obvious. Our data indicate that neither demography, the actual milligrams of DZ as reflected by Body weight, the recent pattern of drinking, nor the family hi~tory of alcoholism explaired the variance. The disagreements in the literature regarding the relationship between ACTH plasma values and benzodiazepine administration probably relate to the dose of the drug used, as extremely high levels of benzodiazepines in animals have been shown to increase both ACTH and cortisol (Pericic et al 1984). Other disagreements could reflect samples that might include subjects with illnesses such as depressive disorder or substance dependence, and the presence of research paradigms that involve stress-inducing manipulations, all factors that can impact on ACTH values (File 1990). Considering the demonstration here that not all subjects have the same amount of change in ACTH following drug infusions, disagreements in the literature probably also reflect the relatively small sample sizes. The results of the present investigation must be viewed in light of a number of methodological constraints. First, only two DZ doses were selected, and each of these yielded only mild-to-moderate levels of intoxication. Thus, a true dose--response curve across a wide range of drug plasma levels could not be established. Second, because determination of hormone levels was only one part of a larger series of evaluations, reflecting time constraints, blood samples could only be taken every 30 rain. Values at 15 min, the time of probable maximal drug effect, would have been especially valuable. In addition, this study was not structured for an adequate evaluation of the mechanisms through which ACTH decreases following DZ. Most investigators speculate that the alterations in ACTH occur as a consequence of hypothalamically regulated changes in CRF release, perhaps in response to a complex interaction between neurotransmitters (Gram and Christensen 1986; Kalogeras et al 1990; Petraglia et al 1986; File 1990). The neurochemical substrates underlying these changes probably involve the benzodiazepine receptors, gamma aminobutyric acid (GABA) activity, and perhaps responses to alterations in levels of norepinephrine and serotonin (Kalogeras et al 1990; File 1990). In this regard, a recent study in rats has demonstrated that the acute administration of benzodiazepines increases hypothalamic CRF concentration while stimulating the release of ACTH (Owens et al 1989). Subsequently, benzodiazepines act at the GABA ionophore regulating paraventricular CRF neurons to inhibit CRF release. In the absence of an appropriate procedure for sampling the hypothalamic secretion of CRF directly, the present data can only be taken to indicate that at the doses of d~:ugs used here there is a marked effect of DZ on ACTH.
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Though not the major focus of this article, this investigation also offered some data consistent with our prior comparisons of sons of alcoholics and controls. The present results show no evidence that sons of alcoholics differ from controls on the pattern or magnitude of ACTH changes following DZ. Though consistent with the prior results on cortisol (Schuckit et al 1991a) as well as those measuring subjective levels of intoxication (Schuckit et al 1991a), the lack of a group differential in response to DZ stands in contrast to the diminished intensity of reaction to alcohol demonstrated in prior samples of sons of alcoholics (Schuckit 1990; Schuck:,~ .~t ai 1987, 1988). The authors thank David Greenblatt for his help in developingthese data. This work was supported by The Veterans Affairs ReseaTchService, NIAAA Grants #05526 and #69012004.
References Calogero AE, Kamilaris TC, Bemardini R (1990): Effects of peripheral benzodiazepine receptor ligands on hypothalamic-pituitary-adrenal axis function in the rat. J PharmacolF.xp Ther 253:729737. De Souza EB (1990): Neuroendocrine effects of benzodiazepines. J Psychiatry Res 24: ! ! 1-119. File SE (1990): Interactions of anxiolytic and antidepressant drags with hormones of the hypothalamie-pituitary-adrenal axis. Pharm,~ol Ther 46:357-375. Gram LF, Christensen P (1986): Benzediazepine suppression of cortisol secretion: A measure of anxiolytic activity? Pharma¢opsychiatry 19:19-22. (3reenblatt DJ, Hermann OR, Lloyd BL (1980): Entry of diazepam and its major metabolite into cerebrospinal fluid. Psychopharmacology 70:89-93. l-lommer DW, Matsuo V, Wolkowitz O, et al (1986): Benzodiazepine sensitivity in normal human subjects. Arch Gen Psychiatry 43:542-551. Kalogera~,~ KT, Calogero AE, Kuribayiashi T, et al (1990): In vitro and in vivo effects of the triazolobenzodiazepine alprazolam on hypothalamic-pituitary-adrenal function: Pharmacological and clinical implications. J Ciin Endocrinol Metab 70:1462-1471. Laakmann G (1984): Effects of psychotmpic drugs (desimipramine, chlorimipramine, sulpiride and diazepam) on the human HPA axis. Psychopharmacology 84:66-70. Lakic N, Pericic O, Manev H (1986): Mechanisms by which picrotoxin and a high dose of diazepam elevate plasma corticosterone level. Neuroendocrinoiogy 43:331-335. Monteiro MG, Schuckit MA, Hauger R, Irwin M, Duthie LA (1990): Growth hormone response to intravenous diazepam and placebo in 82 healthy men. Biol Psychiatry 27:702-710. Owens MJ, Bissette (3, Nemeroff CB (1989): Acute effects of alprazolam and adinazolam on the concentrations of corticotropin-releasing factor in the rat brain. Synapse 4:196-202. Pericie D, Lakic N, Manev H (1984): Effect of diazepam on plasma corticosterone levels. Psy. chopharmacology 83:79-81. Petrag[ia F, Bakalakis S, Facchinetti F, Volpe A, Muller EE, (3enazzani AR (1986): Effects of sodium valproate and diazepam on beta-endorphin, beta-lipotropin and cortisol secretion induced by hypoglycer,ic stress in humans. Neuroendocrinology 44:320-325. Raft H, Findling JW (1989): A new immunoradiometric assay for corticotropin evaluated in normal subjects and palients with Cushing's syndrome. Clin Chem 35:596-600. Risby ED, Hsiao JK, Golden RN, Potter WZ (1989): Intravenous alprazolam challenge in normal subjects: Biochemical, cardiovascular, and behavioral effects. Psychopharmacology 99:508514. Roy-Byme PP, Risch C, Uhde TW (1988): Neuroendocrine effects of diazepam in normal subjects following brief painful stress. J Clin Psychopharmacol 8:331-335.
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Schuckit MA (1990): A prospective study of children of alcoholics. Banbury Report 33:183-194. Schuckit MA, Gold E, Risch C (I 987): Plasma cortisol levels following ethanol in sons of alcoholics and controls. Arch Gen Psychiatry 44:942-945. Schuckit MA, Risch SC, Gold EO (1988): Alcohol consumption, ACTH levels, and family history of alcoholism. Am J Psychiatry 145:1391-1395. Schuckit MA, Hauger RL, Monteiro MG, Irwin M, Duthie LA, Mahler H (1991a): Response of 3 hormones to diazepam challenge in sons of alcoholics and controls. Alcohol Clin Exp Res 15:537-542. Schuckit MA, Duthie LA, Mahler HK, Irwin M, Monteiro MG (1991b): Subjective feelings and changes in body sway following diazepam in sons of alcoholics and control subjects. J Stud Alcohol (in press). Tormey WP, Darraugh AS (1979): The effects of diazepam on sleep, and on the nocturnal release of growth hormone prolactin: ACTH and cortisol. J Clin Pharmacol 8:90--92.
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Adrenocorticotropin Hormone Response to Diazepam in Healthy Young Men Marc A. Schuckit, Richard Hauger, and Jeffery L. Klein
The effects of diazepam (DZ) infusions on changes in adrenocorticotropin hormone (ACTH) are a source of debate. In this study, 66 healthy young adult men were evaluated for changes in plasma ACTH after intravenous inJusions with placebo, 0.12 mg/kg and 0.20 mg/kg of DZ. After the higher DZ dose, 85% of the subjects demonstrated a decrease in ACTH of 20% or more, with the nadir occurring between 30 and 60 min postinfusion and values returning to baseline levels by 180 rain. These results support the conclusion that at the clinically relevant doses used here, DZ infusions are associated with a significant decrease in ACTH. Introduction Studies of the hypothalamic-pituitary-adrenal (HPA) axis have a unique place in medicine and psychiatry. Aberrations in plasma levels and circadian patterns of corticotropin releasing factor (CRF), adrenocorticotropin hormone (ACTH), and cortisol are important in the diagnosis of a number of tumors, they have been studied as potential causes or mitigating factors in major depression, alcoholism, and sleep disorders, and have helped further our understanding of physiological responses to stress (File 1990; De Souza 1990). Our knowledge of the clinical implications and neurochemical controls of the HPA axis has been advanced by the development of systematic perturbations of the system. These include observations of the effects of stress, infusions of hormones such as insulin, and challenges with various medications. The optimal interpretation of results of such studies requires carefully described methods with large enough samples to generate reliable data, because it is unlikely that any single type of challenge will result in a 100% subject response. Reflecting the normal circadian fluctuations, it is also important to control for placebo effects, and for human studies, the challenges must also be safe. F~'lr the latter reason, evaluations of neurohormonai responses to benzodiazepines are attractive. Following such drugs, alterations in cortisol and ACTH are reported to be dose dependent, with increases likely to be observed after very high dose infusions in animals (e.g., 10 mg/kg of DZ), and some indication of decreases in hormone plasma levels after lower doses (e.g., 0.1-1.0 mg/kg) (Lakic et al 1986; File 1990; De Souza 1990; Kalogeras et al 1990). Most studies agree that these lower, more physiological
From the University of California-San Diego. School of Medicine (MAS, RH, JLK) and Alcohol Research Center, Veterans Affairs Medical Center (MAS), San Diego, CA. Received March 5. 1991, revised November 15, 1991. Address reprint requests to Marc A. Schuckit, M.D., Veterans Affairs Medical Center (I J6A), 3350 La Jolla Village Drive, San Diego, CA 92161. © 1992 Society of Biological Psychiatry
0006-3223/92/$05.00
662
B1OLPSYCHIATRY 1992;31:661-669
M.A. Schuckit et al
levels of benzodiazepines attenuate stress-induced or insulin-induced increases in both cortisol and ACTH (Roy-Byrne et al 1988; De Souza 1990; File 1990), and diminish hormone levels in patients with physiological conditions that result in hypersecretion of the HPA axis (Gram and Christensen 1986; De Souza 1990). There is general, but not unanimous, agreement that in the absence of these conditions, physiological doses of a variety of benzodiazepines result in a decrease in cortisol (Hommer et al 1986; Laakmann 1984; Schuckit et al 1991a), but there is more discord regarding changes in ACTH. Some studies indicate a decrease in this hormone similar to that observed with cortisol (Risby et al 1989; Owens et al 1989), but others relate possible increases or no change, or are difficult to interpret data (Tormey and Darraugh 1979; Roy-Byn~e et al 1988; Calogero et al 1990). It is possible that the disparities regarding ACTH might reflect small samples and the variance added by including patients with medical, psychiatric, and substance use problems. Results from samples of 10 or fewer subjects al~o make it difficult to estimate the proportion of individuals who reliably demonstrate a decrease in ACTH following benzodiazepines. In addition, the sensitivity of the usual double-antibody radioimmunoassays for measuring ACTH may not be sufficient to detect inhibition of basal circulating levels. The developmentof a highly sensitive immunoradiometric assay for ACTH now provides a more sensitive method of measuring decreases in circulating ACTH levels below the baseline. The present article presents data on a sample of 6o ,:arefully screened young men for whom ACTH values are available following slow intravenous (iv) infusions of placebo and two clinically relevant doses of DZ.
Methods The 66 men, including 36 who were incorporated in other relevant articles (Schuckit et al 1991a, 1991b; Monteiro et ai 1990) were originally identified between 1985 and 1988 through a structured questionnaire mailed to male students and nonacademic staff at the University of California, San Diego. The subjects were screened further by a subsequent telephone interview with themselves and a resource person when necessary, and with a semistructured personal face-to-face interview. These instruments were used to exclude potential subjects who were alcohol abstainers, whoa met criteria for DSM-III Axis 1 disorders including those related to substance use, or who had medical problems or were taking medications likely to interfere with a drug challenge. As part of a larger investigation, the sample includes subjects who have a biological father who met DSM-III criteria for alcohol abuse or dependence [family history-positive (FHP) men] as well as family history-negative (FHN) controls (Schuckit et al 1991a). After initial selection, subjects were brought to the laboratory for a total of four 9 AM infusions, each separated by a minimum of 72 hr. The men and the laboratory personnel were both blind to the drug condition. The information presented here focuses on data gathered during sessions when subjects were challenged, in random order, with placebo, 0.12 and 0.20 mg/kg of DZ infused over 7 min. The fourth session involved an oral alcohol challenge, generating data not relevant to the hormonal responses to benzodiazepines. The DZ doses were chosen to create intensities of subjective intoxication that are similar to those observed following 0.75 and 1. I ml/kg of ethanol, the doses used historically in our laboratory since the mid-1970s (Schuckit et al 1991a, 1991b). Subjects fasted overnight, arriving in the laboratory at 7:00 AM at which time they were given a light breakfast. Two heparin-locks were inserted into separate antecubital veins in the
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nondominant ann, with one used for the infusion and the other for withdrawal of blood samples. After a minimum of 30 min for acclimation, the infusions were carried out at baseline, and blood samples were withdrawn every half-hour over the 180 rain for analyses of DZ desmethyldiazepam (DMZ), and ACTH plasma levels. During the test sessions, subjects also participated in a series of evaluations of subjective feelings, motor performance, and cognition and, thus, no individual was allowed to fall asleep. Because the protocol included a wide range of tests, time constraints required that blood samples could not be taken more frequently. Blood for ACTH was collected in EDTA vacutainer tubes on ice and separated into plasma by centrifugation at 1000 x g with 90 min of collection. Plasma ACTH concentrations were measured with a highly sensitive and specific immunoradiometric assay that utilizes two antibodies, both with high affinity and specificity for defined regions of the ACTH molecule (Raft and Findling 1989). Following these methods, the highest concentration of ACTH measurable without dilution is approximately 1700 pg/ml, and the limit of detection (assay sensitivity) is 1.0 pg/ml, lntraassay and interassay coefficients of variation are approximately 3% and 7%, respectively. Benzodiazepine plasma levels were determined by an electron-capture gas-liquid chromatography procedure (Greenblatt et al 1980). The major data analytic procedures evaluated ACTH levels following the infusions utilizing a 3 (dose: placebo, low, high) x 6 (hormone sampling time points) repeated measures analysis of variance (ANOVA). The assessment of change during the placebo session was carried out with a single six time point ANOVA, and a subsequent determination of potential differenceg between the reactions to the two different DZ doses was gauged with a multiple points analysis of covariance (ANCOVA), covarying for placebo in a 2 (dose: low, high) x 6 (hormone sampling time points) procedure. Similar analyses were carried out for cortisol. The comparability of the three baseline ACTH levels was evaluated with an ANOVA, revealing no significant difference (F = 0.06; 2, 130 df; p = 0.94); nor were there baseline differences for the subset of men for cortisol (F -0.58; 2, 70 df; p = 0.56). The 66 men were all Caucasian (Schuckit et al 1991a; Schuckit 1990). The average individual was 22. I .4- 1.98 years of age, had completed 15.4 +- I. 18 years of school, weighed 165.1 -+ 22.97 pounds, and consumed alcoholic beverages on 7.6 _ 4.66 days per month, taking 3.2 -+ 1.58 drinks per occasion in the 6 months prior to testing. A drink was defined as 4 ounces of wine, 12 ounces of beer, or 1.5 ounces of 80-proof beverage. No subject had ever used benzodiazepines for a "high." Results During the active challenges, the DZ and DMZ plasma levels peaked by the time of the first blood samples at 30 min, with the diazepam plasma levels returning slowly toward baseline, and the DMZ levels plateauing throughout the experiment, as shown in Table 1. Because of procedural problems, these results were available on only 55 of the 66 men (83%), but are similar to those described in prior publications (Monteiro et al 1990; Schuckit et al 1991a). Figure 1 presents the ACTH values ( -- SEM) at baseline and each half-hour over the 180 min in the three test conditions. Consistent with the time of day during which the samples were gathered, the values during placebo session slowly decreased over time, with an ANOVA revealing a significant time effect (F = 3.83; 5,325 dr; p -- 0.002).
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Table 1. Mean ( "4-SD) for Blood Levels of Diazepam (ng/ml) and Desmethyldiazepam (ng/ml) Following the Active Infusions Dose :
O.12 mg/kg
Time (min)
DZ
30 60 90 120 150 !80
373.5(108.26) 283.5(69.98) 218,6(55,06) 184,0(49,68) 151,5(36.66) 134,2(33.53)
0.20 mg/kg DMZ 15.2(14.92) 16.7(!4 . 4 2 ) i 7,6(14.36) 18.1(14.11) 17,5(13.39) 18,3(!3 . 8 9 )
DZ
DMZ
635.5(124.37) 456.6(98.00) 353. I(90.64) 301.1(74.29) 25I. 3(57.69) 221.7(55.67)
18.6(! I. 32) 22.2(! 1.35) 23.5(12.32) 24.1(11.71) 24.3(! 1.58) 25.2( I 1.55)
In contrast to these results, following both the low-dose and high-dose DZ infusions, there was a rapid and marked decrease in ACTH values by 30 rain, a plateau until 60 min, and then a slow return toward baselim: over the remainder of the experiment. The repeated measures ANOVA considering all three doses and six postbaseline time points revealed a significant dose effect (F = 44.28; 2,130 df; p < 0.0001), and a trend for a main time effect (F ffi 2.07; 5,325 df; p ffi 0.07). There was a significant dose by time interaction (F = 9.04; 10,650; p < 0.0001). Potential differences in ACTH values following low-dose versus high-dose DZ as measured by the ANCOVA covarying for placebo results revealed a trend for a significant dose main effect (F ffi 3.63; 1,65 df; p = 0.06), a significant time main effect (F ffi 15.13; 5,324 df; p < 0.0001), and a nonsignificant dose by time interaction (F = 0.45; 5,325 df; p ffi 0.82). Data on the response of cortisol during similar DZ infusions have been presented elsewhere (Schuckit et al 1991a). Due to budgetary constraints, only 36 of the 66 men
40
3O
E m w L
Figure 1. Plaslaa ACTH response (mean - SEM) to IV infusions of placebo, 0.12 mg/kg DZ, and 0.20 mg/kg DZ over time in 66 healthy men. 10
0
D Plaoebo V Low Dose Olozepam • High Dose Dlozeporn . , , l s i 30 60 90 120 150 Po#t-lnfusTon Time (mlnutee)
! t80
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Table 2. Pearson Correlations of Blood Chemistry Measures Over 180 Min 0.12 mg/kg DZ infusion Correlations for
30
60
90
120
150
180
Summary
DZ with ACTH DZ with Cortisoi DMZ with ACTH DMZ with Cortisol ACTH with Cortisol
-0.11 -0.45 0. I ! -0.09 0.32
-0.11 -0.35 0.08 -0.04 0.06
-0.14 -0.15 0.03 -0.91 0.19
-0.18 -0.15 0.03 0.05 0.22
-0.22 -0.32 0.02 0.00 0.34
-0.13 -0.27 0.03 0.17 0.22
-0.2.~, -0.27 0.00 -0.13 0.41
-0.11 -0.33 0.00 -0.33 0.23
-0.34 -0.23 -0.19 -0.36 0,48
0.20 mg/kg DZ infusion DZ with ACTH DZ with Cortisol DMZ with ACTH DMZ with Cortisol ACTH with Cortisol
-0.28 -0.23 -0.04 -0,07 0.17
-0.20 -0.23 -0.04 -0.07 0.24
-0.05 -0.24 -0.04 -0.16 0.48
-0.12 -0.19 -0.10 -0.21 0.54
-0.11 -0.28 -0.05 -0.33 0.46
reported here also had cortisoi values available, and these have been analyzed in preparation for the evaluation of the relationships of hormone levels to drug plasma levels presented in Table 2. As shown in Figure 2, the decreases in cortisol over time related here are quite similar to our prior report, which had included these 36 men. Regarding cortisol levels following placebo, the ANOVA revealed a significant time effect (F = 13.72; 5,175 df; p < 0.001). For the three doses, the ANOVA revealed significant dose ( F = 17.96; 2,70 df; p < 0.001) and time (F = 24.60; 5,175 df; p < 0.001) main effects, but a nonsignificant dose-by-time interaction ( F = 1.46; 10,350 df; p = 0.16). After covarying for placebo effects in an ANCOVA, the dose main effect was nonsignificant (F = 2.99; 1,35 df; p = 0.09), time was significant ( F = 10.46; 5,174 df;
20
10 16
~ 14
o..
to i
g
R
6 4 2 0
Figure 2. Plasma cortisol response (mean ± SEM) to IV infusions of placebo, 0.12 mg/kg DZ, and 0.20 mg/kg DZ over time in 36 healthy men, a subset of the 65 men in Figure !.
a Placebo V Low Don Dlozepom • High Dose Dlazepom !
DA
30 60 90 t20 150 Poat-lnfuelon Time (mlnutce)
180
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Table 3. Proportion of Subjects with ACTH Decreases of 20% or More from Baseline Following Three Challenges Time Challenge Placebo 0.12 mg/kg DZ 0.20 mg/kg DZ
30 Min (%)
60 Min (%)
27 6! 85
33 79 79
p < 0.001), and the dose-by-time interaction was not significant (F = 0.55; 5,175 df, p = 0.74). These indicate a lack of a significant difference for the two DZ doses on cortisol. The data on cortisol are mentioned here so that the relationship among cortisoi, ACTH, DZ. and DMZ blood levels can be presented on these men in Table 2. The correlations are presented as they relate to the full set of data: 55 men had ACTH and DZ levels available but only 26 had cortisol and DZ. The values are similar if only those 26 men on whom all values were available were used in the analyses. Of course, after both low and high dose challenges, ACTH and cortisol values are positively correlated. Also DZ plasma levels evidence a moderate negative correlation with both ACTH and cortisol across both experimental conditions, with little evidence of differences in the magnitude of these correlations for either the dose or the hormone. DMZ, on the other hand, showed little correlation with ACTH, but a more intense correlation with cortisol, especially after the high dose infusions. A significant change in ACTH following DZ for a sample of 66 men could occur even if a relatively large subgroup demonstrated no alteration in the hormone after the infusion. Unfortunately, the literature is of little help identifying what magnitude of change is clinically relevant. Thus, arbitrarily we evaluated the proportion of men who demonstrated a decrease of between 10% and 30% from baseline values, finding the most prominent differences between placebo and high-dose DZ at a 20% decrement at 30 min. Table 3 demonstrates that although such a change is seen at 30 min in only 27% of the men after placebo, it was observed in 85% after high-dose DZ. An effort was made to determine if the men with such decreases in ACTH after DZ differed from the remainder on relevant va, i hies, but the two groups proved to be similar on all tested characteristics including ago, weight, family history of alcoholism, and their drinking patterns over the prior 6 months. The larger study fi'om which these data were extracted was originally created to determine if sons of alcoholic fathers, themselves at fourfold increased risk for future alcoholism (Schucki~ 1990), differed from well-matched controls on their responses to alcohol and DZ challenges, Though earlier studies have consistently revealed a less intense response for the FHP men following alcohol (Schuckit 1990), there were no indications of a similar phenomenon after infusions of DZ (Schuckit et al 1991a, 1991b). Similar to those earlier results, when the 66 men reported on here were divided into the component 33 FHPs and 33 FHNs, no significant differences nor consistent trends differentiating the family groups were noted for ACTH following any of the three infusion,~. For example, a 3 (dose: placebo, low, high) × 2 (FHP,FHN) x 6 (hormone s~:mpling time points) ANOVA revealed no significant family history main effect (F = 0.99; 1,64 df; p = 0.32), a nonsignificant family history by dose interaction (F = 1.64; 2,128 dr; p =
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0.20), a nonsignificant family history by time factor (F = 1.43; 5,320 df; p -- 0.21), and a nonsignificant family history by dose by time interaction (F = 0.81; 10,640 df; p ffi 0.62). Discussion This study demonstrated that IV infusions of clinically relevant levels of DZ resulted in a rapid decrease in plasma ACTH levels that reversed within 3 hr. These findings were generated in a large sample of healthy young men for whom psychiatric and substance use disorders, medical problems, and recent ingestion of medications were controlled. Even after considering the observation that approximately one-quarter of the subjects demonstrated a 20% or greater decrease in this hormone following placebo, a similar diminution in hormone levels was observed in 85% of the subjects following an active DZ infusion. The reasons for the variability between subjects for the ACTH response to DZ are not obvious. Our data indicate that neither demography, the actual milligrams of DZ as reflected by Body weight, the recent pattern of drinking, nor the family hi~tory of alcoholism explaired the variance. The disagreements in the literature regarding the relationship between ACTH plasma values and benzodiazepine administration probably relate to the dose of the drug used, as extremely high levels of benzodiazepines in animals have been shown to increase both ACTH and cortisol (Pericic et al 1984). Other disagreements could reflect samples that might include subjects with illnesses such as depressive disorder or substance dependence, and the presence of research paradigms that involve stress-inducing manipulations, all factors that can impact on ACTH values (File 1990). Considering the demonstration here that not all subjects have the same amount of change in ACTH following drug infusions, disagreements in the literature probably also reflect the relatively small sample sizes. The results of the present investigation must be viewed in light of a number of methodological constraints. First, only two DZ doses were selected, and each of these yielded only mild-to-moderate levels of intoxication. Thus, a true dose--response curve across a wide range of drug plasma levels could not be established. Second, because determination of hormone levels was only one part of a larger series of evaluations, reflecting time constraints, blood samples could only be taken every 30 rain. Values at 15 min, the time of probable maximal drug effect, would have been especially valuable. In addition, this study was not structured for an adequate evaluation of the mechanisms through which ACTH decreases following DZ. Most investigators speculate that the alterations in ACTH occur as a consequence of hypothalamically regulated changes in CRF release, perhaps in response to a complex interaction between neurotransmitters (Gram and Christensen 1986; Kalogeras et al 1990; Petraglia et al 1986; File 1990). The neurochemical substrates underlying these changes probably involve the benzodiazepine receptors, gamma aminobutyric acid (GABA) activity, and perhaps responses to alterations in levels of norepinephrine and serotonin (Kalogeras et al 1990; File 1990). In this regard, a recent study in rats has demonstrated that the acute administration of benzodiazepines increases hypothalamic CRF concentration while stimulating the release of ACTH (Owens et al 1989). Subsequently, benzodiazepines act at the GABA ionophore regulating paraventricular CRF neurons to inhibit CRF release. In the absence of an appropriate procedure for sampling the hypothalamic secretion of CRF directly, the present data can only be taken to indicate that at the doses of d~:ugs used here there is a marked effect of DZ on ACTH.
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Though not the major focus of this article, this investigation also offered some data consistent with our prior comparisons of sons of alcoholics and controls. The present results show no evidence that sons of alcoholics differ from controls on the pattern or magnitude of ACTH changes following DZ. Though consistent with the prior results on cortisol (Schuckit et al 1991a) as well as those measuring subjective levels of intoxication (Schuckit et al 1991a), the lack of a group differential in response to DZ stands in contrast to the diminished intensity of reaction to alcohol demonstrated in prior samples of sons of alcoholics (Schuckit 1990; Schuck:,~ .~t ai 1987, 1988). The authors thank David Greenblatt for his help in developingthese data. This work was supported by The Veterans Affairs ReseaTchService, NIAAA Grants #05526 and #69012004.
References Calogero AE, Kamilaris TC, Bemardini R (1990): Effects of peripheral benzodiazepine receptor ligands on hypothalamic-pituitary-adrenal axis function in the rat. J PharmacolF.xp Ther 253:729737. De Souza EB (1990): Neuroendocrine effects of benzodiazepines. J Psychiatry Res 24: ! ! 1-119. File SE (1990): Interactions of anxiolytic and antidepressant drags with hormones of the hypothalamie-pituitary-adrenal axis. Pharm,~ol Ther 46:357-375. Gram LF, Christensen P (1986): Benzediazepine suppression of cortisol secretion: A measure of anxiolytic activity? Pharma¢opsychiatry 19:19-22. (3reenblatt DJ, Hermann OR, Lloyd BL (1980): Entry of diazepam and its major metabolite into cerebrospinal fluid. Psychopharmacology 70:89-93. l-lommer DW, Matsuo V, Wolkowitz O, et al (1986): Benzodiazepine sensitivity in normal human subjects. Arch Gen Psychiatry 43:542-551. Kalogera~,~ KT, Calogero AE, Kuribayiashi T, et al (1990): In vitro and in vivo effects of the triazolobenzodiazepine alprazolam on hypothalamic-pituitary-adrenal function: Pharmacological and clinical implications. J Ciin Endocrinol Metab 70:1462-1471. Laakmann G (1984): Effects of psychotmpic drugs (desimipramine, chlorimipramine, sulpiride and diazepam) on the human HPA axis. Psychopharmacology 84:66-70. Lakic N, Pericic O, Manev H (1986): Mechanisms by which picrotoxin and a high dose of diazepam elevate plasma corticosterone level. Neuroendocrinoiogy 43:331-335. Monteiro MG, Schuckit MA, Hauger R, Irwin M, Duthie LA (1990): Growth hormone response to intravenous diazepam and placebo in 82 healthy men. Biol Psychiatry 27:702-710. Owens MJ, Bissette (3, Nemeroff CB (1989): Acute effects of alprazolam and adinazolam on the concentrations of corticotropin-releasing factor in the rat brain. Synapse 4:196-202. Pericie D, Lakic N, Manev H (1984): Effect of diazepam on plasma corticosterone levels. Psy. chopharmacology 83:79-81. Petrag[ia F, Bakalakis S, Facchinetti F, Volpe A, Muller EE, (3enazzani AR (1986): Effects of sodium valproate and diazepam on beta-endorphin, beta-lipotropin and cortisol secretion induced by hypoglycer,ic stress in humans. Neuroendocrinology 44:320-325. Raft H, Findling JW (1989): A new immunoradiometric assay for corticotropin evaluated in normal subjects and palients with Cushing's syndrome. Clin Chem 35:596-600. Risby ED, Hsiao JK, Golden RN, Potter WZ (1989): Intravenous alprazolam challenge in normal subjects: Biochemical, cardiovascular, and behavioral effects. Psychopharmacology 99:508514. Roy-Byme PP, Risch C, Uhde TW (1988): Neuroendocrine effects of diazepam in normal subjects following brief painful stress. J Clin Psychopharmacol 8:331-335.
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Schuckit MA (1990): A prospective study of children of alcoholics. Banbury Report 33:183-194. Schuckit MA, Gold E, Risch C (I 987): Plasma cortisol levels following ethanol in sons of alcoholics and controls. Arch Gen Psychiatry 44:942-945. Schuckit MA, Risch SC, Gold EO (1988): Alcohol consumption, ACTH levels, and family history of alcoholism. Am J Psychiatry 145:1391-1395. Schuckit MA, Hauger RL, Monteiro MG, Irwin M, Duthie LA, Mahler H (1991a): Response of 3 hormones to diazepam challenge in sons of alcoholics and controls. Alcohol Clin Exp Res 15:537-542. Schuckit MA, Duthie LA, Mahler HK, Irwin M, Monteiro MG (1991b): Subjective feelings and changes in body sway following diazepam in sons of alcoholics and control subjects. J Stud Alcohol (in press). Tormey WP, Darraugh AS (1979): The effects of diazepam on sleep, and on the nocturnal release of growth hormone prolactin: ACTH and cortisol. J Clin Pharmacol 8:90--92.
