Urine samples are routinely used in human and animal patients to diagnose health problems; often to investigate or monitor specific health-related problems that essentially may remain silent for extended periods. However, not much work has been performed on crocodilian urine for diagnostics. In general, crocodilian species lack a bladder as a separate storage organ (as found in mammals), possess metanephric kidneys (unable to concentrate urine) and have functional salt excreting glands. Collection of urine from the Nile crocodile (Crocodylus niloticus) is a simple and atraumatic procedure where a dog urinary catheter is used to collect relatively clean urine from the urinary chamber in the crocodile’s cloaca. Unfortunately, in-depth investigations of urine variables, and establishing baseline concentrations, have not been performed on Nile crocodile urine samples before. The specific focus areas of this research project were: (1) determination of urine and plasma biochemical concentrations by means of a standard veterinary clinical pathology profile and the establishment of the ratio between urine and blood biochemical parameters; (2) the validation of a gas chromatographic–mass spectrometric (GC/MS) method for the determination of steroid metabolite concentrations in urine; and (3) using this established analytical method to determine the presence (identify) and concentrations of steroid metabolites in the urine of individual crocodilians.
Urine and plasma samples collected at Izintaba Crocodile Farm during the period November 2005 to July 2006, from captive bred, healthy young Nile crocodiles, were analysed for standard biochemistry variables. The urine samples (n = 101) were analysed for sodium, potassium, chloride, urea, creatinine, calcium, magnesium, phosphate, uric acid, osmolality, and ammonium ion, while the plasma samples (n = 101) were screened for total protein, glucose, sodium, potassium, chloride, urea, creatinine, total calcium, ionised calcium, magnesium, phosphate, uric acid and osmolality. Means, medians and standard deviations were statistically determined, as well as urine to plasma (U/P) ratios for corresponding variables. The value of this project is the establishment of reference concentrations for Nile crocodile urine samples that may become useful for interpretation of laboratory results, in future.
The clinical validation of a GC/MS method for the analysis of urinary steroids in the Nile crocodile was achieved using urine samples from two-year-old Nile crocodiles. The main objective of this investigation was to develop, optimize and validate the laboratory analysis of urinary steroid metabolites. Steroid profiling was performed on individual and pooled Nile crocodile urine samples. Ascending concentrations of representative steroid standards: androsterone, etiocholanolone, dehydroepiandrosterone, 11-OH androsterone, pregnanediol, pregnanetriol, 11-deoxytetrahydrocortisol, tetrahydrocortisone, tetrahydrocortisol and tetra-hydrocorticosterone, were spiked into aliquots of the pooled urine samples, to obtain calibration samples ranging from 0.2 to 20 μg. Sample preparation and analysis methodology were based on a well-established, validated GC/MS method for determination of human urinary steroid metabolites. The validation of the GC/MS method for Nile crocodile urine was successfully completed, by determining lower limits of quantitation and limits of detection for each analyte, obtaining linearity up to the highest calibration level, correlations exceeding 0.90, and recoveries of 82% and more.
Steroid profiling was performed on urine samples collected from a number of mature crocodilian species, namely Nile crocodile, American alligator (Alligator mississippiensis), spectacled caiman (Caiman crocodilus) and dwarf crocodile (Osteolaemus tetraspis). Steroid metabolites were identified and were quantitated and reported per urinary creatinine. Qualitative reporting was conducted in cases where creatinine concentrations were not available. Results included identification and quantitation of the steroid metabolites: androsterone, etiocholanolone, 11-hydroxy androsterone, pregnanediol, pregnanetriol, and the tetrahydro- metabolites of cortisone (THE), cortisol (THF), and corticosterone (THB). In some urinary steroid profiles, several prominent peaks were observed which could not be identified. The study findings confirmed that crocodile urine could successfully be used, as it is commonly used in humans, to determine steroid metabolite profiles. A follow-up study to identify the unknown peaks by structure elucidation with more sophisticated equipment is recommended - this could lead to valuable information about liver metabolism of steroids in crocodilians.
An adrenocorticotrophic hormone (ACTH) stimulation test was conducted on 18 captive Nile crocodiles. The experimental animals were temporarily housed in separate enclosures at Le Croc Crocodile Farm for four weeks, to ensure controlled conditions and easy and frequent access to the animals. Twenty-seven urine samples were collected both pre- and post-ACTH or saline injections. Steroid profiling was performed on 24 of the 27 urine samples to assess the corticosterone and tetrahydrocorticosterone concentrations following the ACTH treatment. Quantitation relative to urine creatinine levels was recorded following analyses with a standardised liquid chromatography/mass spectrometry (LC/MS) method, reporting the concentrations in nmol steroid/μmol creatinine. Unfortunately, a significant increase in urinary corticosterone concentrations 6 h after the injection of Synacthen® (5 μg/kg) was not observed. A possible explanation for this could be that the 6 h period was too short for a significant increase in urinary glucocorticoid metabolite excretion in the Nile crocodile.
In conclusion, this is the first in-depth study that focused, specifically, on Nile crocodile urine for analyses as diagnostic tools and for indices of health. The screening of the urine samples, collected from healthy Nile crocodiles, for a large array of biochemical variables contributed significantly to the database of “normal” concentrations. The establishment of a validated urinary steroid profiling method may significantly contribute to future validation and implementation of innovative diagnostic methods to monitor the health status and endocrine systems of wild Nile crocodiles in Africa.