Frequent Serial Fecal Corticoid Measures from Rats Reflect Circadian and Ovarian Corticosterone Rhythms.

The circadian glucocorticoid rhythm provides important information on the functioning of the hypothalamic-pituitary-adrenal axis in individuals. Frequent repeated blood sampling can limit the kinds of studies conducted on this rhythm, particularly in small laboratories. rodents that have limited blood volumes. and are easily stressed by handling. We developed an assay protocol to measure fecal corticosterone metabolites in repeated samples collected from undisturbed male and female adult Sprague-Dawley rats. This fecal measure provides a non-invasive method to assess changes in corticosterone within a single animal over time, with sufficient temporal acuity to quantify several characteristics of the circadian rhythm: e.g. the nadir, acrophase, and asymmetry (saw-tooth) of the rhythm. Males excreted more immunoreactive fecal corticoids than did females. Across the estrous cycle, females produced more fecal corticoids on proestrus (the day of the preovulatory LH surge) than during estrus or metestrus. These results establish a baseline from which to study environmental, psychological, and physiological. disturbances of the circadian corticosterone rhythm within individual rats.

Figure 1. Base-peak intensity chromatogram of corticosterones loaded and separated on symmetry C18 preconcentration cartridge. Insets are averaged spectra of each chromatographic peak. Analytes: (1) equilin (1,3,5 [10], 7-estratetraen-3-ol-17-one); (2) 1,3,5 [10]-Estratrien-3-ol-17-one; (3) 5a-androstan-17-one; (4) dehydroisoandrosterone (5-androsten-3ß-ol-17-one); (5) androsterone (5a-androstan-3a-ol-17one); (6) progesterone, (7) 11-dehydrocorticosterone (4-pregnen-21-ol-3,11,20-trione); and (8) Corticosterone (4-pregnen-11ß, 21-Diol-3, 20-Dione)

Figure 2. Averaged spectra of female fecal extract analyzed as described above. Ions 373, 367 and 369 correspond to tetrahydro-corticosterone (or 3a, 20a, 21-trihydroxy-5ß-pregnane-11-one), 11-dehydrocorticosterone and 21-hydroxy-5ß-pregnane-3, 11, 20-trione, respectively.

Isolation, Identification, and Quantification of Putative Biochemical Markers Associated with Alcoholism.

There is some evidence for the role of dopamine-derived alkaloids, the tetrahydroisoquinolines (TIQs), in the high alcohol consumption behavior. It has been assumed that biotransformation of ethanol to its active metabolite (acetaldehyde) induces alteration in the metabolism of dopamine and produces TIQs having addictive properties. Salsolinol can be produced in the tissue from the condensation of dopamine with acetylaldehyde with the source of acetaldehyde coming from the oxidation of ethanol. There is evidence that salsolinol levels in the striatum and limbic structures increase following involuntary ethanol consumption in rats. Compared to a control group, alcoholics have been reported to have higher plasma levels of both the R- and S- forms of salsolinol. Conversely, decreased levels of salsolinol were observed in the nucleus accumbens (a dopamine-rich region considered to be involved in regulating alcohol drinking in the P strain of rats relative to the NP strain). This suggests a mechanism in which the P rats may consume alcohol in order to increase the innate salsolinol levels in the accumbens to levels comparable to those seen in ethanol-naïve NP rats.

In order to accurately determine trace levels of salsolinol and other relevant catecholamines involved in alcoholism induction, the needed analytical methodology must involve accurate and reproducible protocols for sample isolation and enrichment from the complex biological mixtures. For the analysis of biological samples, a preliminary purification step is necessary (a) to remove interfering compounds, increasing procedural selectivity, and (b) to extract quantitatively catechols, increasing analysis sensitivity. We are currently developing analytical methodologies to isolate catecholamines present in brain tissue from alcohol-preferring, and non-preferring rats and measure them quantitatively.

Figure 1. Extracted ion chromatogram of catecholamines. Concentration 1 ng/μl, 2 μl loaded on the PBA trap and separated on a reversed-phase C18 column. Order of elution; norepinephrine, epinephrine, impurity, norsalsolinol, L-dopa, and salsolinol.