Value of toxicological investigation in the diagnosis of acute drug poisoning in children.

In the 2 years 1978 and 1979 specimens from 287 children aged between 10 days and 14 years were received for general toxicological investigations. Of the 95 (33%) cases of confirmed poisoning, the diagnosis was established as a direct result of the analyses in 48 patients. No diagnosis was made in at least 85 (30%) of the remaining cases. Benzodiazepines were the drugs most commonly encountered (33%), followed by barbiturates, glutethimide, and meprobamate (15%), salicylate and paracetamol (15%), tricyclic antidepressants (12%), and ethanol (11%). 36 patients were severely poisoned (grade 3 or 4 coma, or convulsions), although only 1 patent died. There was evidence that drug(s) had been administered without authorisation in at least 7 instances, and in 51 (54%) of the poisoned patients there was sufficient concern about the safety of the child or the mode of administration of the drug(s) to institute legal proceedings (8 cases), involve the social services (25 cases), or arrange further medical appointments (18 cases). Drugs are readily available in most households and offer a means of inflicting injury that is less easily detectable than physical assault. For this reason, comprehensive toxicological investigations should be considered in children not only when they may assist in management but also in the presence of unusual or unexplained symptoms which could be drug-induced.

The ontogeny of thymidine kinase in tissues of man and rat.

Thymidine kinase activities, virtually all soluble in rat lung, liver, and small intestine, decreased abruptly late in gestation or immediately after birth. An injection of thyroxine delayed the fetal but not the neonatal changes in liver activity. An injection of cortisol decreased hepatic and pulmonary thymidine kinase activities in both fetal and neonatal rats but had little effect on the intestinal enzyme. Premature extrauterinization led to an earlier occurrence of the quantitative changes in thymidine kinase activity usually seen at term. Birth-associated changes included a rapid transitory increase in the hepatic enzyme and the virtual loss of intestinal thymidine kinase activity. In human tissues, the soluble thymidine kinase in liver remained high between the 11th and 22nd wk of gestation whereas the particulte enzyme, the predominant form in adult liver, rose in the second half of gestation and reached adult levels at birth. In human lung, the soluble enzyme started to decrease by the 16th gestational wk, whereas the particulate thymidine kinase reached the higher adult levels late in gestation. Thymidine kinase in adult human tissues was predominantly particulate.

Relative activities of thymidylate synthetase and thymidine kinase in rat tissues.

The activities of thymidylate synthetase and thymidine kinase were compared in tissues of normal (adult and developing), cortisol-injected, and tumor-bearing rats. The purpose of the study was to determine whether the activities of these two enzymes, which catalyze reactions leading to the same metabolic intermediate, changed proportionately, reciprocally, or independently under different physiological conditions. Both enzymes had high activities in fetal tissues. Thymidine kinase concentrations decreased shortly before or immediately after birth; in several tissues, transient postnatal peaks in thymidine kinase activities appeared within the first 3 weeks after birth. Thymidylate synthetase activities declined gradually after parturition and showed no significant postnatal rises. In sucklings given injections of cortisol, thymidine kinase activities were reduced substantially in eight tissues while thymidlyate synthetase decreased only in lung and thymus of 11-day-old rats. In tumor-bearing rats, thymidine kinase activity increased dramatically in spleen, whereas thymidylate synthetase activities only doubled. In host liver, rises in thymidine kinase activities were not always matched by increases in thymidlyate synthetase. In the tumors, both activities were higher than in most normal adult tissues. Despite the differential sensitivities of the two enzymes to cortisol and tumor bearing, thymidylate synthetase and thymidine kinase were closely correlated in tissues of untreated animals. The Spearman rank correlation coefficient for 112 tissues was 0.895, while the correlation coefficient between the standard scores of the activities was 0.839. The activities of the two enzymes did not appear to be reciprocal or compensatory during normal differentiation or during dedifferentiation associated with tumor bearing, but their potentials for activity were independent of each other.

Uridine kinase activities in developing, adult and neoplastic rat tissues.

Uridine kinase activities were found chiefly in the soluble fractions of rat tissues. In normal adults the activities ranged from 13 munits/g in skeletal muscle to 178 munits/g in colon. Enzyme activities in several rat neoplasms were significantly higher (e.g. in a fibrosarcoma, mammary carcinoma, renal carcinoma, pancreatic carcinoma and lymphocytic lymphoma, but not in a fast-growing Morris hepatoma). The activities were not related to tumour growth rates or sizes. In normal foetal liver, lung, brain, heart and kidney, uridine kinase concentrations equalled or exceeded those in the adult homologous tissue, but maximal activities in liver were reached 3--5 days post partum. In suckling rats the intestinal activity decreased substantially immediately after birth and normally did not rise again until late in the third postnatal week. Premature upsurges could be evoked by an injection of cortisol or by starvation of the pups overnight. Pancreatic activity was absent from 1-day-old rats, and only about 5% of the adult activity was reached by day 20; adult activities were attained rapidly after weaning. In pancreas, precocious formation or uridine kinase was elicited by overnight starvation of 2-week-old rats.

Effects of cortisol or starvation on the activities of four enzymes in small intestine and liver of the rat during development.

The small intestine of the rat, like the liver, is a tissue with high activities of arginase, ornithine aminotransferase, and pyrroline-5-carbozylate reductase. These enzymes are thought to catalyse sequential steps in the synthesis of proline. We have compared the effect of cortisol or brief starvation on the activities of these enzymes and of soluble alanine aminotransrerase in the small intestine and liver during development. In the intestine, cortisol accelerated the increase in arginase activity, reversed the normal 2-week-long post-natal decline in that of pyrroline-5-carboxylate reductase, and delayed the normal decrease, in the third week, of ornithine aminotransferase activity. Starvation of neonates for 18 h raised the activity of arginase slightly, that of pyrroline-5-carboxylate reductase significantly, and had no effect on ornithine aminotransferase activity. Cortisol did not alter the hepatic activities of pyrroline-5-carboxylate reductase in neonates but induced premature rises in the activities of arginase and ornithine aminotransferase. Short starvation did not affect the hepatic activities of any of these enzymes. Alanine aminotransferase activity in both tissues was enhanced by cortisol but not by starvation. Thus in intestine, cortisol elicited some changes in the activity of three functionally related and one unrelated enzyme while starvation evoked changes only in pyrroline-5-carboxylate reductase. Neither stimulus appears to be specific for a metabolic pathway or to trigger a coordinated onset of proline synthesis from arginine.

Glutamate dehydrogenase, alanine aminotransferase, thymidine kinase, and arginase in fetal and adult human and rat liver.

In fetal livers of both man and rat thymidine kinase activity was 12 times higher than in the adult, glutamate dehydrogenase and arginase were present at 20-50% of their adult values, whereas alanine aminotransferase activity was only an insignificant fraction of that in the adult. Although the developmental changes for the four enzymes were quantitatively similar in both species, qualitatively there were some significant differences. In adult human liver, glutamate dehydrogenase activity was distributed almost equally between the cytosol and particles; the concentration of only the soluble enzyme increased after birth. In rat liver, glutamate dehydrogenase remained exclusively a particulate enzyme. The soluble hepatic alanine aminotransferase activity rose in both species after birth (from less than 2 U/g to 41-57 U/g, respectively). Thymidine kinase was wholly soluble in the fetal livers; only in adult human liver was additional activity (at least 50% of the total) found in the particles. Arginase isozymes, identical and apparently the same single isozyme in fetal and adult rat liver, show an ontogenetic change in man. A shift from a single form, common to human fetal liver and fetal kidney, to at least two variants in adult human liver, indicates another complexity of the fully differentiated liver in man.

Enzymes of ornithine metabolism in adult and developing rat intestine.

The levels of 11 enzymes, most of them involved in the metabolism of ornithine, were measured in whole upper intestine, or in duodenum, small intestine and colon of adult rats. The developmental formations in small intestine of arginase, ornithine aminotransferase, and ornithine transcarbamylase were compared with those in liver. Changes with age (late gestation of adult) of the intestinal activities of pyrroline-5-carboxylate reductase, proline oxidase and glutamyl transpeptidase are also described. The results suggest that the proximal part of the intestine is well endowed with enzymes involved in the conversion of ornithine to proline as well as to citrulline. Fetal intestine is rich in proline oxidase and pyrroline-5-carboxylate reductase. The peak levels of ornithine aminotransferase found in intestine in the first 3 postnatal weeks were higher than seen in any other rat tissue. Some of the properties of arginase, ornithine aminotransferase and pyrroline-5-carboxylate reductase in small intestine were compared with those in liver. Isozymes of arginase in small intestine differed from those in liver; the kinetic properties of ornithine aminotransferase were similar in the two tissues. In intestine of 14-day-old rats, the ornithine aminotransferase reaction was reversible, forming ornithine from pyrroline-5-carboxylate. The intestinal pyrroline-5-carboxylate reductase was cold-labile as was the hepatic enzyme in rat.

The heterogeneity of arginases in rat tissues.

Arginase reactions in rat tissues were shown to be catalysed by three isoenzymes which can be separated by bidirectional electrophoresis on polyacrylamide gels. Anodic electrophoresis reveals a migrating band (isoenzyme I) present in all-non-hepatic tissues except submaxillary gland and a non-migrating band found in all tissues. The latter is resolved by cathodic electrophoresis into isoenzymes III (characteristic of liver and submaxillary gland) and a non-moving band (isoenzyme II), present in kidney, intestine and pancreas. Sequential electrophoresis, in the two directions, of mixture of liver and kidney extracts in the same gel columns separated all three isoenzymes. Differences in the solubilization properties, heat-sensitivity and substrate specificity of arginases from different tissues could be correlated with their electrophoretic behaviour. L-Canavanine could replace arginine as substrate in extracts of kidney but not of liver. Both kidney isoenzymes hydrolysed L-canavanine equally well, whereas isoenzyme III from submaxillary gland showed only very low activity. Antiserum against liver arginase interacted with the enzyme with submaxillary gland, but did not inactivate or adsorb arginase from kidney, intestine or pancreas. The distribution of arginase among 16 normal adult rat tissues is presented; the improved, sensitive, assay method was applicable to tissues containing as little as 0.1% of the hepatic activity.

Amino acid metabolizing enzymes in rat submaxillary gland, normal or neoplastic, and in pancreas.

The activities of 12 enzymes, many related to ornithine metabolism, were measured in rat submaxillary gland, submaxillary gland tumors and pancreas. In submaxillary gland, the activities of arginase, ornithine aminotransferase, pyrroline-5-carboxylate reductase and glutamine synthetase were high, but no ornithine transcarbamylase or proline oxidase could be detected. In the fetal submaxillary gland, arginase was at almost adult levels while ornithine aminotransferase reached 50% of its adult value postnatally. Submaxillary tumors deviated from their cognate tissue by lower levels of amino acid metabolizing enzymes and by high concentrations of thymidine kinase. In pancreas, none of the pyrroline-5-carboxylate metabolizing enzymes were as high as in either liver or submaxillary gland. The outstanding activities were those of gamma-glutamyl transpeptidase and glutamate dehydrogenase. Although arginase activities in submaxillary gland and pancreas were quantitatively similar, they differed qualitatively: submaxillary gland contained the same variant as liver while the pancreatic isozymes resembled those of other nonhepatic tissues.

Glutamine synthetase and glutamyltransferase in developing chick and rat tissues.

Concomitant determination of gamma-glutamine-hydroxylamine-glutamyltransferase (GT) and gamma-glutamyl hydroxamate synthetase (GS) activities in chick retina, brain and liver between the 13th day of incubation and a day after hatching showed that while both activities increased late in incubation, in neural tissues their rises were not simultaneous throughout development; in liver both activities were already high on the 14th day of incubation and changed in parallel thereafter. GS and GT activities could be evoked prematurely in all three tissues by cortisol, but GT activity showed higher responses to the hormone. GT and GS were separable by differential solubilization in chick liver but not in retina of chick or rat. The wide spread of the ratios of GT:GS activities in homogenates of a number of chick and rat tissues (1:1 to 73:1) indicates that the GS reaction is not catalyzed by the same protein that catalyzes the GT reactions. Relative amounts of GT(T) (free of GS activity) and of variants of GS (with different competences to catalyze the GT reaction) may govern the changes between the two activities during development and their distribution among different adult tissues in chick and rat.