1. Investigación

The effect of mild stress on various plasma metabolites in the rat has been studied. Mild stress resulted in significant decreases in liver size and glycogen content, as well as in an increase of blood glucose. In addition, plasma lactate, insulin, glycerol and urea, as well as a number of amino acids were altered by stress. These data indicate that minimal stress can have major effects upon the composition of blood, and suggest the need for strict rrecautions on the handling of animals during blood sampling. The site of blood extraction - tail tip i•s. neck - was also found to have a significant effect on plasma lactate. glucosi! and urea concentrations. In stressed animals the differences between tail- and neck blood composition were increased.

A radiochemical method based on dansylation of plasma samples with Dansyl-chloride and thin layer chromatography on polyamide sheets is presented for the quantitative individual determination of plasma amino acids in very small blood samples. The results are first corrected with a norvaline internal standard and secondly with ~pecific recovery factors for each amino acid. The results agree closely with other methods and with already published plasma normal amino acids values in healthy adult rats.

In this work is described a simplified plasma deproteinizatior. procedure using acetone and very small amounts of plasma. The precipitation is carried out in small-bore capillary tubes, and acetone is used as deproteinizing agent. This method can be advantageously compared with heat, Somogyi's, perchloric, and even trichloroacetic deproteinization procedures for its good amino acid preservation and protein removal capabilities.

A method for the determination of the total muscle mass in small experimental animals is presented. Muscle proteins were quantified in the 1 M LiCl-soluble and distilled water-insoluble fraction of the eyeless, brainless, eviscerated and skinned carcass, as compared with a striated muscle sample from the same animal used as standard and processed in the same way as the whole carcass. The non-muscular tissues left in the carcass do not interfere with the final results. The total muscle mass in adult rats was estimated as 42.0 ± 2.8 % of the ln vivo weight.

Se discuten las expresiones de actividad enzimatica mas utilizadas en estudios comparados de actividad en diferentes tejidos: microkatales por unidad de peso del tejido, por unidad de peso de proteina y por unidad de peso de AON. Se utiliza tambien la expresi6n de microkatales presentes en un 6rgano determinado referidos a unidad de peso del animal, 100 g en el caso de la rata. Las diversas expresiones se han aplicado a los niveles de aspartato transaminasa, glutamato deshidrogenasa y AMP desaminasa en higado, musculo estriado de pata trasera y riiiones de rata adulta. De Ios datos presentados se concluye que las mediciones de actividades enzimaticas en tejidos deben ser expresadas en mas de una forma, ya que la informaci6n obtenida a partir de una sola de ellas puede ser substancialmente distinta de la obtenida con otra de ellas, dando lugar a posibles conclusiones err6neas de! papel metab6lico jugado por el enzima en un tejido determinado.

A 24-h fast induced different patterns of change in the amino acid concentrations of liver, muscle, plasma and blood cells. Starvation produced generalized increases in blood amino acids despite decreases in plasma, thus increasing the blood cells amino acid pool. Muscle increased amino acid levels with fasting, while the changes were much more buffered in liver. The fraction of essential amino acids carried by the blood was considerably greater than that of muscle and liver. The size of muscle pool in the whole rat was much greater than that of liver and more than two orders of magnitude higher than that of the whole blood. Fasting-induced changes agree with the known transport of amino acids from muscle and other peripheral tissues towards the liver and other splanchnic organs.

Rats chronically treated with two daily doses of tolbutamide, glibenclamide or glipentide were compared with animals treated with placebo. Plasma individual amino acids were determined at 0, 3, 7, 10, 12, 14, 17, 24, 27 and 29 days of treatment 16 hours after the administration of the drug. Rats were fasted for 48 h periods at days 10 to 12 and 27 to 29 of the experiment. Sulfonylurea treated animals show minor changes in the plasma aminogram, although glipentide and glibenclamide produced greater effects than tolbutamide. At the 3rd day after the onset of the treatment, plasma levels of glutamate+ glutamine, arginine and histidine appeared significantly reduced in glipentide and glibenclamide treated animals. When plasma samples were collected 3 h after the drug administration at the 24th day of treatment, the only observed change was a decrease in the levels of arginine in the glipentide treated animals. Fasting produced decreases in plasma levels of alanine, pro line, cysteine, tyrosine, methionine +ornithine and tryptophan, there were no changes in serine, aspartate + asparagine, threonine citruline, arginine and lysine; and glycine, glutamate+ glutamine and leucine + isoleucine show increases. These changes were rapidly compensated with refeeding, appearing a "rebound effect" in certain amino acids. Both fasting and refeeding affect very little the effect of sultonylureas on plasma amino acid levels, although for some individual amino acid they reduce or enhance the effect of the fasting. These small effect of sulfonylureas on plasma amino acid levels could be the result of the juxtaposition of different factors, including the effects of these drugs on circulating insulin levels, on protein biosynthesis and amino acids transamination and hepatic gluconeogenesis.