American Journal of Primatology 13:325-332 (1987) BRIEF REPORT Effects of Anesthetic Agents on the Adrenocortical System of Female Baboons MARGARET L. WALKER1, GERALD J. PEPE3, NELSON L. GARNET?, AND EUGENE D. ALBRECHT' 'Departments of Obstetricd@necology and Physiology, University of Maryland School of Medicine, Baltimore, 'Central Animal Facility, University of Maryland School of Medicine, Baltimore, 3Department of Physiology, Eastern Virginia Medical School, Norfolk, Virginia Invasive surgical procedures are often used to study the reproductive and adrenocortical endocrine systems in primates. Anesthetic agents must, therefore, be used that have the least confounding effects on these systems. The present study was designed to characterize various adrenocortical endocrine responses of female baboons (Papw anubis), each treated for 120 minutes with an infusion of ketamine HC1 (6 mg/min) in 5% dextrose in water (0.40 mumin), a combination of ketamine and acetylpromazine (0.6 mg acetylpromazine and 6 mg ketamine HCllmin) in 5%dextrose in water, or inhalation of vaporized halothane (1.0% halothane, NzO 25%, 1litedmin; 0 2 75%, 3 liters/min). Blood samples were collected throughout the treatment period, and serum was assayed for prolactin (PRL), dehydroepiandrosterone (DHA), dehydroepiandrosterone sulfate (DHAS), and cortisol (F).No significant elevations in DHA, F, or PRL concentrations were found following infusion of ketamine alone. Only serum DHAS concentrations were significantly altered after long-term exposure to ketamine. Acetylpromazine increased PRL concentrations tenfold to levels significantly greater than those in ketamine- and halothane-treated animals but had no effect on serum DHA, DHAS, or F. Treatment with halothane had no effect on serum PRL, DHA, or DHAS but did suppress F (>40%) concentrations over time. These data indicate that ketamine is best suited for the collection of biological samples when deep analgesia is not required but that halothane is preferable in the latter situation. Key words: cortisol, prolactin, dehydroepiandrosterone, ketamine, acetylpromazine, halothane INTRODUCTION The baboon is an excellent nonhuman primate model for the study of reproductive endocrinology because the patterns of hormone production and metabolism are relatively similar to those in humans [Goldzieher & Axelrod, 1969;Albrecht & Pepe, 1985;Pepe & Albrecht, 1985al. Invasive experimental procedures are often employed Received February 2, 1987; revision accepted April 14, 1987. Address reprint requests to Dr. Eugene D. Albrecht, Department of Obstetrics and Gynecology,University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, MD 21201. 0 1987 Alan R. Liss, Inc. 326 I Walker et a1 in our studies with baboons to determine the factors essential to the regulation of the hypothalamic-hypophyseal-adrenocorticalaxis, including the formation of cortisol @'), dehydroepiandrosterone (DHA), and DHA sulfate (DHAS). Because the endocrine system is sensitive to the differential effects of various anesthetics [Ferin et al, 1976; Quadri et al, 1978; Puri et al, 19811, it is essential that anesthetic agents are used that produce the fewest possible confounding effects. Previous studies [Albrecht et al, 19771 with baboons have demonstrated that ketamine prevents the stress-induced rise in F typically associated with handling and restraint. However, ketamine alone apparently does not provide the analgesia required for major abdominal surgery [Hughes & Lang, 19831. To circumvent this problem, a combination of ketamine and a central nervous system depressant such as acetylpromazine is commonly employed to induce the analgesidanesthesia required for major surgery. Similarly, general anesthesia achieved by inhalation of halothane is an effective means of attaining the necessary state of analgesia. The present study was designed to determine the effects of ketamine, the combination of ketamine and acetylpromazine, and halothane on the adrenocortical hormones F, DHA, and DHAS in baboons. In addition, the influence of these anesthetics on prolactin (PRL), a hormone elevated by stress [Aidara et al, 19811 and recently shown to regulate adrenal gland function [Pepe & Albrecht, 1985b], was assessed. Elucidation of the effects of these anesthetics on endocrine parameters has important implications for the study of baboon reproductive physiology. MATERIALS AND METHODS Subjects Six adult female baboons (Papio anubis) weighing 10-12 kg were studied at random times of the menstrual cycle. The animals were maintained in an environmentally controlled animal facility in which food (Purina Monkey Chow 5037, Ralston-Purina, St. Louis, MO) and fruit were provided twice daily and water was available ad lib. Each animal was used as its own control and was treated with the anesthetic agents described below in a randomly assigned fashion to control for carry-over effects. A minimum of seven days elapsed between anesthetic treatments. Procedures Between 1000 and 1100 hours, baboons were briefly restrained in their cages for initial sedation with 100 mg ketamine HC1 Parke, Davis & Co., Detroit, MI), which was administered intramuscularly. Blood (5.0 ml) was immediately withdrawn from a peripheral saphenous vein to serve as the time 0 sample. The animals were immediately transferred to a surgical suite and subjected to one of the following regimes for 120 minutes. Ketamine HCl. Six animals were placed on their left side and maintained under anesthesia with ketamine HC1 (6 mg/min; 0.40 ml/min) in 5% dextrose in water infused with an I-Med 922 infusion pump (I-Med Corp, San Diego, CA) via a catheter (Bard 1917R, 24 inches) inserted into the saphenous vein. Ketamine HC1-acetylpromazine. In six baboons, 1.0 ml of acetylpromazine (10 mg/ml; Aveco Co., Inc., Fort Dodge, IA) was added to 9.0 ml ketamine (100 mg/mU, and this solution was constantly infused via the saphenous vein at a rate of 6 mg ketamine-0.6 mg acetylpromazine/min (0.40 ml/min) in 5% dextrose in water. Halothane. Following ketamine sedation, four of the six baboons were placed in a supine position and intubated with an endotrachial tube through which a mixture of vaporized halothane (Fluothane, Ayerst Laboratories, NY) and other gases (NzO, 25%, 1 literlmin; and 02,75%, 3 literdmin) were passed. Between 0 and 30 minutes only, 1.5%halothane and 0 2 were administered. From 30 minutes Endocrine Response to Anesthesia in Baboons I 327 until termination of treatment, the concentration of the halothane was reduced t o 1.0% in the presence of N20 carrier. Blood samples (5.0 ml) were collected at 5, 10, 15, 30, 45, 60, 75, 90, 105, and 120 minutes during anesthesia. Samples were kept on ice and centrifuged, and serum was stored at -20°C until assay. Assays Concentrations of F, DHA, and DHAS were measured by using radioimmunoassays described previously [Pepe et al, 1977; Townsley & Pepe, 19771. The cortisol assay had a sensitivity of 31.0 pg/ml. Intra- and interassay coefficient of variation (CV) were assessed by using serum pools and averaged 8.0 and 9.1% respectively. The sensitivity of the DHA assay was 16.0 pg/ml, and the intra- and interassay CV were 8.2 and 9.8% respectively. The DHAS assay could detect 41 pg/ml with intraand interassay CV of 6.1 and 10.2% respectively. PRL concentrations were determined by using a double antibody RIA kit developed by Diagnostic Products Corp (Los Angeles, CAI. The antiserum was generated against porcine PRL. The ability of this antibody to detect baboon PRL was validated by measuring serum PRL concentrations in a female baboon infused for 60 minutes with thyroid-releasing hormone (TRH; 3 pgkg body weight). Within ten minutes of TRH infusion, PRL concentrations rose from a baseline of 8.5 ng/ml to 108 ng/ml, representing a tenfold increase. The PRL assay was sensitive to 5 ng/ml and the assay is optimized for linearity throughout the 5-200 ng/ml range. Analysis of Data The effects of anesthetic agent on hormone concentrations were evaluated for each hormone by using analysis of variance with repeated measures (sampling times) and a nested variable (anesthetic). This statistical evaluation permitted determination of the main effect of anesthetic, the pattern of hormone change over time, and the interaction between time and anesthetic. Effects were considered significant at P < .05. Post hoc comparisons were made by using Newman-Keuls (N-K) procedure [Keppel, 19821. Data are expressed as mean SE. RESULTS Serum PRL Concentrations Basal (ie, time 0) serum PRL concentrations in animals subsequently treated with ketamine, ketamine-acetylpromaine, and halothane were 8.4 0.8,7.4 +_ 3.6, and 7.2 + 3.6 ng/ml respectively. The administration of ketamine-acetylpromazine elevated serum PRL t o concentrations significantly above those achieved with halothane (N-K, P < .Ol) and ketamine (N-K, P < .01; Fig. 1)alone. Treatment with ketamine-acetylpromaine increased PRL, from a baseline of 7.4 +_ 3.6 ng/ml to a maximum at 120 minutes of 79.5 2 18.0ng/ml, which represented a tenfold increase in serum PRL concentrations. Serum F Concentrations Basal serum F concentrations (pg/lOO ml) were similar in ketamine (17.9 _+ 1.5)-, ketamine-acetylpromaine (18.4 1.4)-,and halothane (21.9 & 2.0)-treated animals respectively (Fig. 2). Treatment with halothane depressed F concentrations slightly, to 13.1 +_ 2.1 pg/100 ml at 120 minutes (N-K, P < .05).However, serum F was not significantly affected by ketamine or ketamine-acetylpromaine. Serum DHA and DHAS Concentrations Basal serum DHA concentrations were 10.6 +_ 2.3, 10.3 & 2.3, and 10.2 k 4.0 ng/ml in animals treated with ketamine, ketamine-acetylpromaine, and halothane 328 I Walker et a1 15 0 30 45 60 75 90 105 120 MINUTES Fig. 1. Concentrations (mean 5 SE) of peripheral serum prolactin (PRL) in female baboons receiving ketamine (0---O), ketamine-acetylpromazine (@-@), or halothane (A-A) anesthesia over a 120minute period. 1 0 . 1 1 15 1 1 1 1 1 1 x) 45 60 75 90 105 1 120 MINUTES Fig. 2. Concentrations (mean k SE) of serum cortisol (F) in female baboons receiving ketamine (0---O), ketamine-acetylpromaine (0-01, or halothane (A-A) anesthesia over a 120-minute period. Endocrine Response to Anesthesia in Baboons I 329 E )a: " E 10- -* Y -- I n 0 15 30 45 60 75 90 105 120 MINUTES Fig. 3. Concentrations(mean f SE) of serum dehydroepiandrosterone(DHA) in female baboons receiving ketamine (0---O), ketamine-acetylpromazine (0-O), or halothane (A-A) anesthesia over a 120minute period. respectively. Serum concentrations of DHA were not significantly changed by the anesthetic agents (Fig. 3). Serum concentrations of DHAS are depicted in Figure 4. At time 0, baseline DHAS concentrations in animals treated with ketamine, ketamine-acetylpromaine, and halothane were 28.5 I 8.0, 30.2 k 6.8, and 28.5 k 11.4 pg/lOO ml respectively. Serum concentrations of DHAS were significantly elevated (N-K, P < .01) after 75 minutes of treatment with ketamine alone and continued to rise throughout the treatment period, reaching levels 100% above baseline. Treatment with ketamine plus acetylpromazine appeared to increase DHAS concentrations throughout the final 60 minutes of treatment; however, this increase was not significant. Halothane treatment had no effect on serum DHAS concentrations. DISCUSSION In the present study, the effects of three commonly used anesthetics on the serum concentrations of hormones typically measured in the study of the hypothalamic-hypophyseal-adrenocorticalaxis were determined in baboons. Serum concentrations of PRL, which characteristically are elevated by stress [Aidara et al, 19811, were rapidly and strikingly elevated by acetylpromazine, a centrally acting dopamine receptor blocker. Because dopamine is a putative PRL-inhibitoryfactor [Meites & Clemens, 19721, inhibition of the dopaminergic system results in increased PRL release. A well-established relationship exists between hyperprolactinemia and altered reproductive function in which elevated PRL inhibits luteinizing hormone release directly [Williams et al, 19791 or via diminished gonadotropin-releasing hormone secretion [Nakano et al, 19751. Furthermore, Pepe & Albrecht [1985b] recently have shown that PRL regulates adrenal androgen production in fetal/ neonatal baboons. Therefore, when studying endocrinological events such as menstrual cyclicity, gonadotropin secretion, regulation of the adrenal gland, or any physiological response affected by PRL, acetylpromazine does not appear to be a suitable anesthetic. Ketamine alone, when administered iv continuously, had no effect on serum PRL, F, or DHA concentrations in baboons. In contrast, others [Quadri et al, 1978; Aidara et al, 1981;Puri et al, 19811have reported that ketamine increased PRL and F in various cercopithecines. Methodological or species differences may account for 330 / Walker et al 0 15 30 45 60 75 90 105 120 MINUTES Fig. 4. Concentrations (mean k SE)of serum DHA sulfate @HAS) in female baboons receiving ketamine (0---O), ketamine-acetylpromazine (0-01, or halothane (A-A) anesthesia over a 120-minute period. these apparently discrepant findings. For example, Quadri et a1 [19781 administered a single injection of ketamine and found that PRL concentrations increased within 30 minutes but returned to baseline by 60 minutes; Puri et a1  reported that only multiple injections of ketamine increased serum PRL concentrations. In the present study, serum concentrations of DHAS only, the most abundant adrenal androgen produced during baboon pregnancy [Townsley & Pepe, 1977; Walker et al, 19871 were elevated by long-term treatment with ketamine alone. This effect appears to be confined to the sulfated form of the adrenal androgen DHA and does not appear t o be mediated by either PRL or ACTH, because neither PRL nor F concentrations were affected by ketamine treatment. Caution should be used, therefore, when studies of the metabolism of DHAS requiring long-term sedation of the animal are performed. Halothane, an alternative to ketamine-acetylpromaine, decreased slightly the serum concentrations of F, a steroid hormone that originates primarily from the adrenal cortex; but halothane had no significant effect on DHA and DHAS. This decline in peripheral serum adrenocortical steroid may have reflected a decrease in production and/or an increase in the metabolic clearance rate of this steroid. Halothane produced no effect on serum concentrations of PRL in baboons, despite reports to the contrary in rhesus monkeys [Quadri et al, 19781. Halothane administered to immobilized animals increased PRL concentrations above those found during immobilization alone, yet PRL was also significantly elevated as a result of immobilization alone [Quadri et al, 19781. Therefore, it is possible that immobilization, and not halothane, may have been the primary stimulant of PRL release in these animals. Overall, the effects of halothane on the hormones examined in the present study were relatively slight, particularly when compared to those observed with Endocrine Response to Anesthesia in Baboons I 331 acetylpromazine.However, when coupled with reports that halothane may adversely affect blood chemistry (eg, gases and acid-basebalance) and other regulatory mechanisms [Goosen et al, 19841, we suggest that caution should be used and results interpreted carefully when using halothane in nonhuman primates. In summary, ketamine alone had no influence on the serum concentrations of PRL, F, or DHA and produced a rise in DHAS only after 75 minutes of administration. Ketamine therefore appears to be an excellent dissociative agent for the study of hormones important in primate reproductive physiology. However, the ability of ketamine to produce analgesia to visceral pain is suspect, and it is no longer considered a suitable anesthetic agent for use in major surgery [Hughes & Lang, 19831. As surgical intervention is often required to study definitively the regulation of the endocrine system, alternative anesthetic agents are required. The present study indicates that halothane had relatively minor effects on hormones of the adrenocortical system. It would appear that in baboons, ketamine is well suited for the collection of biological samples when deep analgesia is not required, but that halothane anesthesia is preferable in the latter situation. CONCLUSIONS 1. In baboons, ketamine administration had no effect on serum PRL, DHA, or F concentrations. 2. Long-term exposure (>60 minutes) to ketamine significantly elevated serum concentrations of DHAS. 3. The dopamine antagonist acetylpromazine significantly elevated serum PRL concentrations in baboons. 4. Halothane slightly decreased serum F concentrations. 5 . In baboons, ketamine was best suited for the collection of biological samples when deep analgesia was not required, and halothane anesthesia was preferable when deep anesthesia was necessary. ACKNOWLEDGMENTS The authors would like to thank Ms. Susan J. Larsen and Mr. Richard J. Roh for their expert technical assistance. This work was supported by NIH grants R01 HD-13294 and F32 HD 06903. REFERENCES Aidara, D.; Tahiri-Zagret, C.; Robyn C . Serum prolactin concentrations in mangabey (Cercocebus atys lunulatus) and patas (Erythre cebus patas) monkeys in response to stress, ketamine, TRH, sulpiride and levodopa. JOURNAL OF REPRODUCTION AND FERTILITY 62~165-172,1981. Albrecht, E.D.; Nightingale, M.S.; Townsley, J.D. Stress-induced decreases in the serum concentration of progesterone in the pregnant baboon. JOURNAL OF ENDOCRINOLOGY 77:425426,1977. Albrecht, E.D.; Pepe, G.J. Regulation of progesterone and estrogen formation during Laboon pregnancy. P p 175-199 in R E SEARCH IN PERINATAL MEDICINE tIV) PERINATAL ENDOCRINOLOGY.E.D.'A~brecht; G.J. Pepe, eds. Ithaca, Perinatology Press, 1985. Ferin, M.; Carmel, P.W.; Warren, M.P.; Himsworth, R.L.; Frantz, A.G. Phencyclidine sed- ation as a technique for handling rhesus monkeys: Effects on LH, GH and prolactin secretion. PROCEEDINGS OF THE SOCIETY FOR EXPERIMENTAL BIOLOGY AND MEDICINE 151:428433,1976. Goldzieher, J.W.; Axelrod, L.R. Urinary metabolites of 4-14C progesterone in the baboon (Pupio sp.). GENERAL COMPARATIVE ENDOCRINOLOGY 13:201-205. 1969. Goosen, D.J.; Davies, J.H.; Maree, M.; Dormehl, I.C. The influence of physical and chemical restraint on the physiology of the chacma baboon (Pupio ursinus). JOURNAL OF MEDICAL PRIMATOLOGY 13:339351,1984. Hughes, H.C.; Lang, C.M. Control of pain. Pp 207-216 in ANIMAL PAIN PERCEPTION AND ALLEVIATION. R.L. Kitchell; H.H. Erickson, eds. Bethesda, American Physiological Society, 1983. 332 I Walkeret a1 Keppel, G. DESIGN AND ANALYSIS: A RE- Puri, C.P.; Puri, V; Anand Kumar, T.C. SEARCHERS HANDBOOK. Englewood Serum levels of testosterone, cortisol, proCliffs, Prentice-Hall, 1982. lactin and bioactive luteinizing hormone in Meites, J.; Clemens, J.A. Hypothalamic conadult male rhesus monkeys following cagetrol of prolactin secretion. VITAMINS AND restraint or anaesthetizing with ketamine HORMONES 30:165-221,1972. hydrochloride. ACTA ENDOCRINOLONakano, R.; Kayashima, F.; Mori, A.; KotGICA (COPENHAGEN) 97:118-124,1981. suji, P.; Hashiba, V.; Tojo, S. Ovarian re- Quadri, S.K.; Pierson, C.; Spies, H.G. Effects sponse to exogenously administered human of centrally acting drugs on serum prolactin gonadotropins during the postpartum pelevels in rhesus monkeys. NEUROENDOriod. AMERICAN JOURNAL OF OBSTETCRINOLOGY 27:136-147,1978. RICS AND GYNECOLOGY 121:187-192, Townsley, J.D.; Pepe, G.J. Serum dehydro1975. epiandrosterone and dehydroepiandrostePepe, G.J.; Albrecht, E.D. Transplacental rone sulfate in baboon (Pupio papio) pregcorticosteroid metabolism during baboon nancy. ACTA ENDOCRINOLOGICA pregnancy. Pp 201-217 in RESEARCH IN 85~415-421. 1977. PERINATAL MEDICINE (IV)PERINATAL Walker, M.L.; Pepe, G.J.; Albrecht, E.D. ENDOCRINOLOGY. E.D. Albrecht; G.J. Changes in the pattern of androgen formaPepe, eds. Ithaca, Perinatology Press, 1985a. tion in vitro by the baboon fetal adrenal Pepe, G.J.; Albrecht, E.D. Prolactin stimugland at mid and late gestation. BIOLOGY lates adrenal androgen secretion in infant OF REPRODUCTION, (1987, in press). baboons. ENDOCRINOLOGY 117:1968- Williams, R.F., Johnson, D.K.; Hodgen, G.D. 1973,1985b. Resumption of estrogen-induced gonadotroPepe, G.J.; Titus, J.A.; Townsley, J.D. Inpin surges in postpartum monkeys. JOURcreasing fetal adrenal formation of cortisol NAL OF CLINICAL ENDOCRINOLOGY from pregnenolone during baboon (PapiopaAND METABOLISM 49:422-428,1979. pio) gestation. BIOLOGY OF REPRODUCTION 17:701-705,1977.