American Journal of Primatology 60:161–166 (2003) BRIEF REPORT Diurnal Patterns of Urinary Steroid Excretion in Wild Chimpanzees MARTIN N. MULLERn and SUSAN F. LIPSON Department of Anthropology, Harvard University, Cambridge, Massachusetts Urinary testosterone and cortisol concentrations were quantified in a large number of samples (4500) collected from wild male chimpanzees (n ¼ 11) over the course of 1 year. For both steroids, urinary concentrations were higher and more variable in the morning than in the afternoon. Urinary creatinine levels showed no such diurnal pattern. These patterns are consistent with studies of steroid excretion in humans and gorillas. This study emphasizes the importance of considering time of day as a confounding variable in field studies of primate endocrine function. It also suggests that if a small number of samples are to be used to characterize an individual’s basal steroid levels, afternoon samples may be preferable because they show less intra-individual variability. Am. J. Primatol. 60:161–166, 2003. r 2003 Wiley-Liss, Inc. Key words: testosterone; cortisol; urinary steroids; chimpanzees; diurnal pattern INTRODUCTION Detailed human studies have shown that circulating cortisol levels exhibit a predictable diurnal pattern, peaking between the morning hours of 0600 and 0800, and steadily declining to a nadir at B0100 hr [Van Cauter, 1990]. This circadian rhythm is endogenously driven by the central nervous system [Liotta & Krieger, 1990], and its synchronization is influenced by both light/dark and sleep/ wake cycles [Morin & Dark, 1992]. Although few studies on this subject have been conducted on primates in the wild, preliminary data suggest that both Old World monkeys (e.g., Macaca fascicularis [van Schaik et al., 1991]) and apes (e.g., Gorilla gorilla [Czekala et al., 1994; Robbins & Czekala, 1997]) conform to the human pattern of diurnal cortisol production [Whitten et al., 1998]. Testosterone production in human males shows a similar, but bimodal, diurnal pattern [Van Cauter, 1990]. Testosterone production reaches its zenith Contract grant sponsor: U.S. National Science Foundation; Contract grant numbers: SBR-9729123; SBR-9807448; Contract grant sponsor: L.S.B. Leakey Foundation. n Correspondence to: Martin N. Muller, Department of Anthropology, University of Michigan, Ann Arbor, MI 48109. E-mail: firstname.lastname@example.org Received 21 October 2002; revision accepted 19 June 2003 DOI 10.1002/ajp.10103 Published online in Wiley InterScience (www.interscience.wiley.com). r 2003 Wiley-Liss, Inc. 162 / Muller and Lipson between 0400 and 0800 hr, wanes throughout the day, effects a modest recovery between 1600 and 1800 hr, and drops to a nadir at B2400 hr. The etiology of this rhythm remains enigmatic, because it is usually, but not always, associated with diurnal variation in LH secretion [Van Cauter, 1990; Matsumoto, 2001]. Its adaptive significance is not currently understood. The few studies that have examined temporal variation in primate testosterone production indicate there are potential differences between hominoids and Old World monkeys [Whitten et al., 1998]. While gorillas match the human pattern of high morning and low evening levels [e.g., Robbins & Czekala, 1997], many Old World monkeys show peaks of testosterone secretion between 2100 and 2400 hr, and an early-morning nadir [Whitten et al., 1998; Dixson, 1998]. The ultimate explanation for these differences is not clear. Minor fluctuations in both testosterone and cortisol secretion are superimposed on the diurnal rhythms described above. Between 12 and 24 times per 24 hr, the testes release a pulse of testosterone in response to the pulsatile release of LH from the pituitary, and GnRH from the hypothalamus [Van Cauter, 1990]. On average, cortisol exhibits seven to nine such spikes in 24 hr, in response to the episodic release of ACTH [Liotta & Krieger, 1990]. The pulsatile nature of steroid secretion complicates the use of plasma measurements to estimate basal hormone titers, since circulating levels of steroid can fluctuate significantly over a matter of minutes [e.g., Weick, 1981]. The use of urinary assays mitigates this problem, however, because excreted steroid represents an average of circulating levels between urinations [Lasley et al., 1994]. Because there is a time lag between steroid release and steroid excretion, urinary levels of hormone reflect endocrine status several hours prior to sampling [Whitten et al., 1998]. Urinary cortisol, for example, lags approximately 2–4 hr behind plasma cortisol in humans and chimpanzees [Pollard, 1995; Bahr et al., 2000]. In this study we examined concentrations of immunoreactive testosterone and cortisol in a large number (4500) of urine samples collected from wild chimpanzees in Kibale National Park, Uganda. The aim of the study was twofold: first, to test whether chimpanzees show significant diurnal fluctuation in steroid excretion that could affect the design of studies relating social behavior to endocrine function, and second, to determine whether, in light of such variability, morning or afternoon samples may be preferable for characterizing an individual’s basal steroid levels. METHODS Between January and December 1998, one of the authors (M.M.) collected urine samples from 11 adult male chimpanzees (16–44 years old) at the Kanyawara study site in Kibale National Park, Uganda. These males were well habituated to observers, and could be followed without disturbance. First-morning urine samples were regularly collected from the chimpanzees, who predictably urinate upon waking. Samples were also collected opportunistically throughout the day. When a chimpanzee urinated from a tree, the urine was trapped in a disposable plastic bag attached to a 2-m pole. If a bag could not be placed in time, the urine was pipetted from leaves in the ground layer of vegetation. Immediately after collection, the identity of the chimpanzee, the date, and the time of urination were recorded. To minimize the risk of sample cross-contamination, urine was collected from vegetation only when it was clear that multiple individuals had not urinated in Steroid Excretion in Wild Chimpanzees / 163 the same area. Care was also taken to avoid collecting urine contaminated with feces. One to 24 hours after collection (mean: 6.5 hr), the urine samples were processed and stored in a propane-powered freezer that consistently maintained a temperature between –181C and –231C. Frozen samples were transported on both ice and dry ice to Harvard University, where we performed all hormone analyses. Steroid levels were quantified by radioimmunoassay according to published protocols [Lipson & Ellison, 1989] adapted for use with primate urine. Before they were assayed, the urine samples were deconjugated by hydrolysis. First 100 ml of urine were combined in a test tube with 20 ml of the enzyme b-glucuronidasearylsulfatase and 300 ml of pH 5 buffer. This mixture was then incubated overnight in a 371C water bath. The testosterone assay is based on a four-position tritiated competitor (Amersham-Searle, Arlington Heights, IL) and an antiserum raised against testosterone-11-BSA (#250; provided by Gordon Niswender, Colorado State University). This antiserum has reported cross-reactivities of 46% with DHT, and 17% with androstenedione and dihydroepiandrosterone. A detailed HPLC analysis by Hagey and Czekala  indicated that testosterone produces the dominant (59%) immunoreactive peak for this antibody with chimpanzee urine. Hydrolyzed urine samples were extracted twice in diethyl ether prior to assay, with recoveries individually monitored by the addition of trace amounts of tritiated T. Recoveries averaged 90%. Separation of bound and free steroid after a 24-hr incubation at 41C was accomplished by adsorption of free steroid to dextrancoated charcoal. The bound competitor was measured in a RackBeta liquid scintillation counter. Quality control was maintained by monitoring values of urine pools at three different levels. Assay sensitivity, the least amount distinguishable from 0 with 95% confidence, averaged 11,000 pmol/L. Intra-assay variability (CV) at the 50% binding point of the standard curve was 6.6%. Interassay variability averaged 6.6%, 6.2%, and 6.4% for high, medium and low pools, respectively (n ¼ 17). Linearity of response was verified by assaying serial dilutions of both testosterone standard (predicted vs. observed values: r2 ¼ 1, Po0.0001) and chimpanzee urine (predicted vs. observed values: r2 ¼ 0.99, Po0.0001). The cortisol assay is based on a four-position tritiated competitor (Amersham-Searle) and an antiserum raised against cortisol-3-0-carboxymethylether-BSA (#07-121016; ICN Biomedicals, Irvine, CA). This antiserum has reported cross-reactivities of 11.4% with 21-desoxycorticosterone, 8.9% with 11desoxycortisol, and 1.6% with corticosterone. Cross-reactions with other naturally occurring steroids are nonsignificant. The details of the cortisol assay are similar to those of the testosterone assay, except that the urine samples were not hydrolyzed and the steroid was not extracted with ether. Cortisol was assayed directly from unpurified urine diluted 1:10 with distilled water. For the cortisol assay, sensitivity averaged 5,250 pmol/L. Intra-assay variability (CV) at the 50% binding point of the standard curve was 6%. Interassay variability averaged 7.2%, 6.7%, and 15.6% for high, medium, and low pools, respectively (n ¼ 16). Linearity of response was verified by assaying serial dilutions of both cortisol standard (predicted vs. observed values: r2 ¼ 0.99, Po0.0001) and chimpanzee urine (predicted vs. observed values: r2 ¼ 0.99, Po0.0001). To correct for variation in urine concentration, steroid levels were indexed to creatinine [Lasley et al., 1994], which was quantified colorimetrically using the Jaffee reaction [Taussky, 1954]. 164 / Muller and Lipson RESULTS When 522 urinary testosterone measurements from 11 adult male chimpanzees were plotted against collection time, a statistically significant decline in immunoreactive testosterone was apparent throughout the day (r2 ¼ 0.14, Po0.0001, n ¼ 522). However, because some males contributed more samples than others to the data set, it is possible that this relationship may have been affected by sampling bias. To examine this, we divided the day into 14 1-hr intervals (0500–1900 hr) and calculated, for each hour, an individual mean testosterone level for each chimpanzee male. We then took the average of these individual means to get a ‘‘mean of individual means’’ for each hour. These means of individual mean testosterone levels showed a clear decline throughout the day (r2 ¼ 0.83, Po0.0001, n ¼ 14, Fig. 1a). Similar patterns emerged when immunoreactive testosterone concentrations from the most frequently sampled male (LK) were analyzed separately (all LK samples: r2 ¼ 0.29, Po0.0001, n ¼ 101; hourly means: r2 ¼ 0.5, P ¼ 0.007, n ¼ 14). When 505 urinary cortisol measurements from 11 adult male chimpanzees were plotted against collection time, there was a statistically significant decline throughout the day (r2 ¼ 0.22, P ¼ 0.0001, n ¼ 505). When means of individual mean cortisol were calculated for each hour, these also showed a significant decline throughout the day (r2 ¼ 0.85, Po0.0001, n ¼ 14, Fig. 1b). Similar patterns emerged when immunoreactive cortisol measurements from the most frequently sampled male (LK) were analyzed separately (all LK samples: r2 ¼ 0.29, Po0.0001, n ¼ 93; hourly means: r2 ¼ 0.69, Po0.001, n ¼ 14). Paired comparisons between mean male concentrations of immunoreactive testosterone in morning (before 1000 hr) and afternoon (after 1000 hr) samples indicated significantly higher concentrations in the morning samples (morning: mean ¼ 599, SD ¼ 162; afternoon: mean ¼ 416, SD ¼ 80; Wilcoxon signed rank test, Z ¼ 2.58, P ¼ 0.01, n ¼ 11 males). The same was true for immunoreactive cortisol (morning: mean ¼ 404, SD ¼ 77; afternoon: mean ¼ 200, SD ¼ 59; Z ¼ 2.934, P ¼ 0.003, n ¼ 11 males). Mean morning creatinine levels, on the other hand, did not differ significantly from afternoon levels (morning: mean ¼ 0.57, SD ¼ 0.08; afternoon: mean ¼ 0.62, SD ¼ 0.13; Z ¼ 0.8, P ¼ 0.42, n ¼ 11 males). Furthermore, when creatinine values from 540 samples collected from 11 adult males were plotted against collection time, the association between the two was negligible (r2 ¼ 0.04, Po0.0001, n ¼ 540). Finally, when means of individual mean creatinine were calculated for each hour, no significant relationship between the two was observed (r2 ¼ 0.04, P ¼ 0.52, n ¼ 14). Intra-individual variation in steroid excretion was higher in the early morning than in the afternoon. In the three most frequently sampled males, the standard deviations (SDs) for samples from the first three collection hours (0500–0800) were considerably higher than those from the final three collection hours (1600–1900) for both immunoreactive testosterone (a.m./p.m. SDs ¼ 190/60, 241/138, 312/121) and cortisol (a.m./p.m. SDs ¼ 266/45, 154/77, 252/148). DISCUSSION This study represents the largest set of urinary steroid values collected from apes in the wild. The results suggest that urinary concentrations of immunoreactive testosterone and cortisol in male chimpanzees are generally higher and more variable in the morning than in the afternoon. This pattern is consistent with plasma, salivary, and urinary measurements of these steroids in humans [Van Cauter, 1990; Dabbs, 1990; Czekala et al., 1994]. In the present study, few Steroid Excretion in Wild Chimpanzees / 165 Fig. 1. Steroid excretion and time of day. a: When means of individual mean male testosterone levels are calculated across 14 1-hr intervals, a statistically significant decline is evident throughout the day (r2 ¼ 0.83, Po0.0001, n ¼ 14). b: The same is true for cortisol (r2 ¼ 0.85, Po0.0001, n ¼ 14). Error bars show standard error of the mean. samples were collected after 1800 hr, so it was not possible to determine whether male chimpanzees, like humans [Van Cauter, 1990], exhibit a secondary afternoon peak in circulating testosterone. As expected, there was no evidence for an effect of time of day on excreted creatinine. This study emphasizes the importance of considering time of day as a confounding variable in field studies of primate endocrine function. It also suggests that if a small number of samples are to be used to characterize an individual’s basal steroid levels, afternoon samples may be preferable because they show less intra-individual variability [see also Czekala et al., 1994]. This may be particularly important for studies of social behavior and steroid production. 166 / Muller and Lipson For example, while we found a positive and significant correlation between afternoon testosterone levels and dominance rank among the adult male chimpanzees at Kanyawara, the same relationship did not exist for morning samples [Muller & Wrangham, in press]. ACKNOWLEDGMENTS For sponsoring long-term research in Kibale National Park, we thank the Uganda Wildlife Authority, Makerere University Biological Field Station, and Richard Wrangham. For assistance in the field we thank John Barwogeza, Christopher Katongole, Francis Mugurusi, Donor Muhangyi, Christopher Muruuli, Peter Tuhairwe, Michael Wilson, and Ross Wrangham. We are grateful to Peter Ellison and Cheryl Knott for providing laboratory facilities, and to Ross Wrangham for assistance in the laboratory. This research was supported by grants to Martin Muller from the U.S. National Science Foundation (awards SBR-9729123 and SBR-9807448) and the L.S.B. Leakey Foundation. REFERENCES Bahr NI, Palme R, Mohle U, Hodges JK, Heistermann M. 2000. Comparative aspects of the metabolism and excretion of cortisol in three individual nonhuman primates. Gen Comp Endocrinol 117:427–438. Czekala NM, Lance VA, Sutherland-Smith M. 1994. Diurnal urinary corticoid excretion in the human and gorilla. Am J Primatol 34:29–34. Dabbs JM. 1990. Salivary testosterone measurements: reliability across hours, days, and weeks. Physiol Behav 48:83–86. Dixson AF. 1998. Primate sexuality: comparative studies of the prosimians, monkeys, apes, and human beings. New York: Oxford University Press. Hagey LR, Czekala NM. 2003. Comparative urinary androstanes in the great apes. Gen Comp Endocrinol 130:64–69. Lasley BL, Mobed K, Gold EB. 1994. The use of urinary hormonal assessments in human studies. Ann N Y Acad Sci 709:299–311. Liotta AS, Krieger DT. 1990. ACTH and related peptides. In: Baulieu EE, Kelly PA, editors. Hormones: from molecules to disease. New York: Chapman and Hall. Lipson SF, Ellison PT. 1989. Development of protocols for the application of salivary steroid analyses to field conditions. Am J Hum Bio1:249–255. Matsumoto AM. 2001. The testis. In: Felig P, Frohman LA, editors. Endocrinology and metabolism. New York: McGraw Hill. p 635–706. Morin LP, Dark J. 1992. Hormones and biological rhythms. In: Becker JB, Breedlove SM, Crews D, editors. Behavioral endocrinology. Cambridge: MIT Press. p 473–504. Muller MN, Wrangham RW. Dominance, aggression and testosterone in wild chimpanzees: a test of the ‘‘challenge hypothesis.’’ Anim Behav (in press). Pollard TM. 1995. Use of cortisol as a stress marker. Am J Hum Biol 7:265–274. Robbins MM, Czekala NM. 1997. A preliminary investigation of urinary testosterone and cortisol levels in wild male mountain gorillas. Am J Primatol 43:51–64. Taussky HH. 1954. A microcolorimetric determination of creatine in urine by the Jaffe reaction. J Biol Chem 208:853–861. Van Cauter E. 1990. Diurnal and ultradian rhythms in human endocrine function: a minireview. Horm Res 34:45–53. van Schaik CP, van Noordwijk MA, van Bragt T, Blankenstein MA. 1991. A pilot study of the social correlates of levels of urinary cortisol, prolactin, and testosterone in wild long-tailed macaques (Macaca fascicularis). Primates 32:345–356. Weick RF. 1981. The pulsatile nature of luteinizing hormone secretion. Can J Physiol Pharmacol 59:779–785. Whitten PL, Brockman DK, Stavisky RC. 1998. Recent advances in noninvasive techniques to monitor hormone–behavior interactions. Yearb Phys Anthropol 41:1–23.