RESUMO
The validation of the analytical technique for the determination of polyamines in cerebral tissue using HPLC based on o-phthalaldehyde post-column derivatization is described. The polyamines were separated in a LiChrospher100 RP18 column. Elution gradient was formed with two mobile phases: A (sodium acetate 0.1 M + sodium octanesulphonate 0.01 M, pH = 4.5) and B (sodium acetate 0.2 M + sodium octanesulphonate 0.01 M)/acetonitrile (10:3), pH = 4.5) in a 1.2 ml/min flow rate. The derivative eluent was monitored by fluorescence (excitation, 345 nm; emission, 455 nm). Besides excellent linearity (putrescine, r = 0.9816; spermidine, r = 0.9920; spermine, r = 0.9901), the technique demonstrated intra and inter-day precision (< 20 percent) as well as recovery (spermidine = 92.56 percent; spermine = 84.47 percent). Quantification limits were 0.22 pM for putrescine, 76.44 pM for spermidine and 51.44 pM for spermine. The method demonstrated to be robust, simple and highly reproducible for polyamine determination in tissues.
A validação técnica analítica para determinação de poliaminas em tecido cerebral utilizando cromotografia líquida de alta eficiência (HPLC) e derivação pós-coluna com o-ftaldialdeído é descrita. A separação das poliaminas deu-se em coluna LiChrospher 100 RP18. O gradiente de eluição foi formado por duas fases móveis A (acetato de sódio 0,1M + octanossulfonato de sódio 0,01 M) e B (acetato de sódio 0,2 M + octanossulfonato de sódio 0,01 M)/acetonitrila (10:3), fluxo de 1,2 ml/min. O eluente foi monitorado por fluorescência (excitação, 345 nm; emissão, 455 nm). Além da excelente linearidade (putrescina, r = 0,9816; espermidina, r = 0,9920; espermina, r = 0,9901), a técnica demonstrou adequada precisão intra e interdia (< 20 por cento) e recuperação (espermidina = 92,56 por cento; espermina = 84,47 por cento). Os limites de quantificação foram 0,22 pM para putrescina, 76,44 pM para espermidina e 51,44 pM para espermina. O método demonstrou ser consistente, simples e altamente reprodutível para a determinação proposta.
RESUMO
The present study investigated the effects of protein malnutrition and environmental stimulation on biochemical and behavioral parameters in rats. The concentrations of polyamines in the frontal cortex, striatum, and hippocampus, as well as plasma corticosterone levels, were measured. The exploratory behavior was analyzed using the elevated plus-maze (EPM) test. Rats received either 16 percent (well-nourished - W) or 6 percent (malnourished - M) protein diets and were divided into stimulated or non-stimulated groups. Malnutrition increased corticosterone levels and decreased plasma protein and anxiety. Non-stimulated rats tested in the EPM had increased corticosterone levels and decreased frontal cortex, striatum and hippocampus protein and polyamines contents. Stimulation decreased open arm entries in the EPM in M animals and increased closed arm entries in the W ones. Stimulation increased frequency and time spent in risk-assessment behaviors. These results suggest that both malnutrition and EPM testing are distressing situations, as indicated by increased corticosterone levels. These results are consistent with lower anxiety and/or higher impulsiveness in M animals.