The metabolic fate of dietary terpenes in folivorous marsupials

The metabolic fate of two commonly occurring dietary terpenes in Australian marsupial eucalypt folivores has been studied. The detoxification mechanisms employed by these folivores were considered from an ecological perspective. Interactions between herbivores and their respective diets has received considerable attention over the last quarter of a century. A popular hypothesis by Freeland and Janzen (1974) states that feeding behaviour of herbivores is influenced by limitations of the body's mechanisms for detoxifying plant secondary metabolites (PSM). However, very little is understood about the physiological processes and limitations of detoxification in wild animals. This study provides an insight into these processes in marsupial eucalypt folivores from a range of feeding niches. An interspecies comparison was made of the metabolism of a bolus dose of the monoterpene p-cymene in a generalist herbivore, the brushtail possum (Trichosurus vulpecula), and three specialist folivores, the greater glider (Petauroides volans), the ringtail possum (Pseudocheirus peregrinus) and the koala (Phascolarctos cinereus), as well as in the laboratory rat (Rattus norvegicus) which provided a direct comparison to published data. Each animal was dosed, intragastrically, with single doses of p-cymene. Urine and faeces were collected pre- and post-dose. Chronic ingestion of 1,8-cineole, emulating its natural occurrence in the diet of brushtail possums and koalas, was also studied. Possums were fed an artificial diet in which 1,8-cineole concentrations were manipulated to cover a range of concentrations while koalas were fed Eucalyptus cephalocarpa, which has a terpene profile dominated by 1,8-cineole. Metabolites were identified by extraction, gas chromatography and mass spectrometry. Standards for many metabolites were isolated from urine to allow quantitation. A novel p-cymene metabolite, 2-p-carboxyphenylpropan-1,2-diol, was identified in the koala. Twelve.novel 1,8-cineole metabolites were identified from brushtail possum and koala urine. Eleven of these were isomers of hydroxy cineolic acid. The isomeric structure and partial stereochemistry for 7-hydroxy-9-cineolic acid were determined by NMR and mass spectrometry. Observational data from chronic ingestion of 1,8-cineole in possums demonstrated a threshold in maximum daily intake in most possums between 3 and 4 g/kg. The pattern of metabolites excreted in the brushtail possum during chronic ingestion demonstrated induction of enzymes responsible for producing the more oxidised metabolites during the first days of ingestion. There was no evidence of saturation of metabolic pathways associated with the larger intakes of 1,8-cineole. For both terpenes, species-specific patterns of metabolite excretion were evident and reflected the natural occurrence of eucalypt leaves in the different diets. The number and degree of oxidation of metabolites, as well as the role of conjugation, varied depending on the natural feeding behaviour of the animals. To facilitate studying the pattern of metabolism, terpene metabolites were grouped according to the total number of oxygen atoms (up to four) acquired during oxidation. Overall there was a progressive increase in the proportion of more extensively oxidised metabolites; from the rat, to the generalist and through to the specialist folivOres. The generalist utilised a multiplicity of non-specific oxidative pathways producing an array of metabolites covering all degrees of oxidation (9 metabolites of p-cymene and 18 metabolites of 1,8-cineole). On the other hand, the specialists had high capacity and specific oxidative pathways resulting in relatively few metabolites, all of which were radically oxidised. Glucuronidation was important in the generalists, compensating for their inability to excrete the same degree of radically oxidised metabolites as specialists. Approximately 40 - 50 % of p-cymene metabolites and up to 60 % of 1,8-cineole metabolites were conjugated with glucuronic acid in brushtail possums. No significant conjugation of metabolites was observed in the specialists. Increased polarity, whether achieved by glucuronidation or extensive oxidation, presumably results in the same overall enhanced capacity to excrete metabolites. It is proposed that, for specialists, oxidative efficiency reduces the necessity for subsequent conjugation, conserving glucuronic acid, a valuable resource in a nutritionally limited diet. The brushtail possum, however, consumes a varied and arguably better quality diet and can afford to excrete glucuronic acid in the detoxification of terpenes. Given that PSMs, such as terpenes, are always present in the eucalypt leaf diet of these folivores, these different strategies indicate that adaptation of detoxification mechanisms vary with dietary specialisation.