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Diurnal patterns of urinary steroid excretion in wild chimpanzees.

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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: mnmuller@umich.edu
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 [2003] 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.
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