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.

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.

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.

Plasma amino acid concentrations, together with other metabolic parameters were determined in thyroidectomized rats treated with daily injections of sal ins (hypothyroid), and 250 ,ug/kg L-T4 {hypothyroid). Data were compared with sham-operated controls. TherP. is a genera! incre'!SE> in plasma amino acid concentrations in hyperthyroidism, a limited in· crease only in several amino acid r.oncentrations in hypothyroid rats as compared with controls, and a considerable difference between the plasma aminograms of both groups. Amino acid homeostasis seems to be subject to greater modification in hyperthyroidism than in hypothyroidism.

The effects of food deprivation for up to 24 hours on plasma metabolic parameters in the rat have been studied. Liver dry weight and glycogen content dropped significantly from a hours of food deprivation onwards. Total muscle glycogen supplied about as much glycosyl residues or precursors as did the liver. Plasma glucose, urea, lactate and total and essential amino acids decreased significantly from 3 hours of fasting onwards. Glycerol, free fatty acids, beta-hydroxybutyrate and acetoacetate showed significant increases with fasting. Alanine, serine, arginine, threonine, aspartate plus asparagine and proline showed significant decreases with fasting. Several other amino acids showed almost nc change with fasting. Lysine, leucine plus isoleucine and taurine showed biphasic changes in their concentrations with a minimum at 6 hours and a transient recovery at 12 hours of fasting. Essential amino acids decreased more than the non essential ones. With fasting there is a shift in ammonia disposal with lower urea concentrations as nitrogen is better conserved. The results seem to suggest that there i~ a constant release of substrates, through liver and peripheral tissue proteolysis, that is counteracted by differential utilization of amino acids during fasting.

Female rats were treated with two daily equihypoglycemic doses (as observed in acute treatment) of tolbutamide, glibenclamide or glipentide . by stomach tube, and were compared with control ammals treated with the suspending medium alone. On day 29 the rats were subjected to a 48 h fast and then were injected intraperitoneally with 100 .μMoles of C' -alanine. Blood samples were collected before and 5, 15 and 30 minutes after the alanine injection, at which time the animals were killed. Blood glucose levels increased after the injection of alanine in all groups, but at the different times stll:died, both the glibenclamide and glipentide treated_ anu_n~ls showed hypoglycemia versus controls. The rad10act1:"1ty found in blood glucose and liver glycogen and glycendeglycerol decreased in the glibenclamide treated anima~s compared with controls while in the other groups 1t was similar. The increase in liver glycogen after the injection of alanine was also diminished in the_ glibenclamide treated animals. Alanine produced an mcrease in the plasma levels of gluconeogenic, basic, aromatic and sulphur-containing amino acids in the controls, while in the animals treated with glipentide the alanine effect was less pronounced. The results show an impairment of gluconeogenic function in glibenclamide treated animals. The effects of both tolbutamide and glipentide were less dramatic. Nevertheless, the findings hinted at an effect of both drugs upon glycogen metabolism in liver.

The effect of ethyl ether, pentobarbital sodium (Nembutal®) and chloral hydrate anesthesia on the concentrations of glucose, lactate, glycerol, insulin, individual amino acids and urea have been studied in rat plasma, as well as the glycogen content in the animal liver and striated muscle. Control animals were injected with saline solution. Two samples were taken from each animal at 15 and 30 minutes from the beginning of anesthesia. These samples were obtained from the cut tail's tip and from the neck wound produced by beheading the animal. Results show considerable differences between the three sets of animals and their controls as well as in their tail versus neck values. The overall effect of the three anesthetics used were roughly the same in spite of their different actions upon the different parameters studied. This was most marked in the aminograms. Significant changes induced were rather similar in number for all. The metabolite concentration profile in each situation may be used as a reference for possible artifacts induced by any experimental approach using these anesthetics.