Methodological confounds of measuring urinary oxidative stress in wild animals

Biomarkers of oxidative stress (OS) are useful in addressing a wide range
of research questions, but thus far, they have had limited application to
wild mammal populations due to a reliance on blood or tissue sampling. A
shift towards non-invasive measurement of OS would allow field ecologists
and conservationists to apply this method more readily. However, the
impact of methodological confounds on urinary OS measurement under field
conditions has never been explicitly investigated. We combined a
cross-sectional analysis with a field experiment to assess the impact of
four potential methodological confounds on OS measurements: 1) time of
sampling, 2) environmental contamination from foliage; 3) delay between
sample collection and flash-freezing in liquid nitrogen; 4) sample storage
of up to 15 months below -80C. We measured DNA oxidative damage
(8-hydroxy-2’-deoxyguanosine, 8-OHdG), lipid peroxidation
(malondialdehyde, MDA), Total Antioxidant Capacity (TAC) and uric acid
(UA) in 167 urine samples collected from wild Zanzibar red colobus
(Piliocolobus kirkii). We found that MDA was higher in samples collected
in the morning than in the afternoon but there were no diurnal patterns in
any of the other markers. Contamination of samples from leaf surfaces and
time frozen at -80°C for up to 15 months did not affect OS marker
concentrations. Freezing delay did not affect OS levels cross-sectionally,
but OS values from individual samples showed only moderate to good
consistency and substantial rank order reversals when exposed to different
freezing delays. We recommend that the impact of storage time on OS marker
concentrations and diurnal patterns of OS markers be considered when
designing sampling protocols. However, given the high stability we
observed for four OS markers subject to a variety of putative
methodological confounds, we suggest that urinary OS markers provide a
valuable addition to the toolkit of field ecologists and conservationists
within reasonable methodological constraints.