Joseph Krasner

Studies on reduction of azo-linkages in human placental homogenates

Abstract Investigations of the capacity of human placental tissue to metabolize exogenous substrates revealed a system present in the soluble fraction of placental homogenates which increased the NADPH-dependent reduction of the azo-linkage of neoprontosil. Characterization of the enzyme system involved indicated striking differences in comparison to the azo-reductase system(s) present in hepatic tissues. Activity of the placental system was not affected by additions in vitro of magnesium ion, nicotinamide, NADP or NAD, and was only very slightly inhibited when incubated under atmospheres of pure oxygen of carbon monoxide. Placental azo-linkage reduction was enhanced by addition in vitro of EDTA, reduced glutathione and glucose 6-phosphate. The addition in vitro of flavins resulted in a decreased rate of conversion of neoprontosil to sulfanilamide. Flavin mononucleotide appeared to possess the greatest inhibitory effect. The system was also inhibited by sulfhydryl reagents. Comparisons of specific activities from homogenates of tissue obtained early in the gestational period vs. that obtained at term revealed no statistically significant differences. Studies on the mechanism of increased azo-linkage reduction revealed that an enzymicnonenzymic system involving d -glucose 6-phosphate: NADP oxidoreductase (EC 1.1.1.49) and 6-phospho- d -gluconate: NADP oxidoreductase (EC 1.1.1.44) could account for this increase in azo-linkage reduction.

VARIATIONS IN DETOXICATION ENZYMES DURING MAMMALIAN DEVELOPMENT

It has been repeatedly demonstrated that the developing fetus and newborn infant are more sensitive than the adult to the effects of many pharmacologic agents.Q Differences in absorption, distribution, and excretion may be present in the newborn organism, but the principal factor in this response differential appears to be variation in the rates of detoxication. Drug metabolic processes have been studied in vitro in several animal species and, in most instances, activities are lower in the newborn organism compared with the a d ~ l t . ~ . ~ Variations in drug metabolism appear to be genetically determined and the enzymic activity observed at any given age is dependent upon the species and strain of organism as well as the substrate employed in the reaction. Our primary objective in our initial studies was to determine how soon after birth one could detect and differentiate the contribution to drug metabolism made by genetic endowment. To study drug response, hexobarbital was selected because its pharmacologic effect, sleeping time (loss of righting reflex), is easily measurable, its metabolism has been carefully studied, and the correlation between pharmacologic effect and biotransformation of the drug is fairly well established. In the following studies5 the hypnotic effect of hexobarbital was measured as the sleeping time or time interval in minutes from injection of the drug until restoration of the righting reflex. The drug was administered i.p. (0.7% (w/v) aqueous solution) in a dosage of 50 or 100 mg/kg except in the newborn, for whom the S.C. route was used. To maintain body temperature, the newborn mice were kept in a ventilated, warm room at 35°C. Hypnotic investigations of all other ages were carried out at room temperature (2626OC). Animals were used only once for the experimental studies because of the well-documented increase in drug-metabolizing ability which is known to accompany prior administration of barbiturates. The mean values for sleeping time in adult mice following i.p. injection of 100 mg/kg of hexobarbital are shown in FIGURE 1. Twelve animals of each sex and strain were used in the assay procedure. The actual values (with standard errors of the mean) in the male animals are as follows: &BL, 18.70k0.96 minutes; Ralb/C, 33.64+ 1.28 minutes; 1295, 61.67 f 5.70 minutes. The differences between the strains are statistically significant at the P < .01 level. Within any given strain there was remarkably little variation in hypnotic response in animals of the same sex and age, but in all strains tested there was a distinct male sex preponderance with the male sleeping more than twice as long as his female counterpart. This sex difference was of considerable interest since it suggested a hormonal mediation of drug response. In the C5, BL strain where detailed inquiry was made, sleeping times in the female were correlated with the estrus cycle determined by vaginal smear. The shortest sleeping times (4.93 It 1.9 minutes) in this strain occurred during the phase of estrus and proestrus, when estrogenic effect is known to be greatest. Sleeping

Effect of Intrauterine Growth Retardation on the Activities of Fetal Intestinal Enzymes in Rats

The activities of maltase, lactase, alkaline phosphatase and enterokinase were followed in the small intestine of rats during prenatal development. These enzymes were detectable only after the 17th day of gestation. Furthermore, each enzyme exhibited a different pattern of prenatal presence. Maltase activity appeared first (day 18), followed by lactase and alkaline phosphatase (day 19) and then enterokinase (day 20). Except for enterokinase, all of the enzymes attained a level of activity close to the newborn levels at the final day of gestation. Induced intrauterine growth retardation during the 3rd trimester led to a decrease in intestinal weight proportional to the reduction of body weight. These decrease in size of the small intestine was caused by a reduction in cell number rather than cell size. Induced intrauterine growth retardation also resulted in a selective reduction in the specific activities of lactase and alkaline phosphatase, but not of enterokinase and maltase. These results suggest that reduction in maternofetal blood flow in the 3rd trimester of gestation will cause a selective decrease in some brush border enzymes (lactase and alkaline phosphatase) but does not effect others (maltase and enterokinase).

Developmental changes in mouse liver alcohol dehydrogenase

Abstract Alcohol dehydrogenase activity in the supernatant fraction of liver homogenates was studied in adult and newborn mice. Newborn mice were found to have a lower specific activity than adult animals. K m values were higher in the new born animal. Electrophoresis showed two bands for the adult and only one for the newborn, with a greater intensity seen in adult.

Postnatal Developmental Changes in Hepatic Bilirubin UDP-GIucuronyl Transferase

Activity of bilirubin glucuronyl transferase in mouse liver homogenates is low at birth, reaches a peak at 14 days of age, 2–3 times greater than adult values. Kinetic studies showed marked difference

Small Intestinal Epithelial Brush Border Enzymatic Changes in Suckling Mice Infected with Reovirus Type 3

Summary: Suckling mice infected with reovirus type 3 were examined for changes in the epithelial brush border of the small intestine. After 3 days of infection with reovirus type 3, no significant changes were found in intestinal morphology or activity of any enzymes tested. After 6 days, villi were shortened and blunted with lymphangiectatic lesions and mild mononuclear infiltration in the lamina propria. In addition, there was a significant decrease in lactase (P < 0.001) and enterokinase activity (P < 0.05). However, there were no significant changes in the activities of alkaline phosphatase. In contrast, maltase (P < 0.001) and leucine amino-peptidase (P < 0.05) activities in the infected mice were significantly increased. These data suggest that brush border enzymes are affected differently by reovirus infection.Speculation: The decrease in lactase activity and increase in maltase and leucine aminopeptidase in infant mice infected with reovirus type 3 can be a reflection of accelerated maturation subsequent to an increased turnover of the epithelial cells of the crypt and villi or to an induction of specific enzymes. In addition, it is possible that the severe decrease of lactase activity in comparison to other brush border enzyme changes is due to the fact that lactase is more vulnerable than are other enzymes to mucosal injury and the possibility that lactase is a receptor for the virus.

Isolation of biliverdin from amniotic fluid.

Abstract A procedure for the isolation of biliverdin in amniotic fluid is described. Identification of the isolated pigment is made by comparing standards using spectrophotometry and thin-layer chromatography.

Reovirus type 3 infection in a suckling mouse: the effects on pancreatic structure and enzyme content.

Summary: Alterations in pancreatic function and structure were examined in suckling mice infected intraperitoneally with reovirus type 3. The results were compared to pancreatic zymogen enzyme activities and histology in adult mice infected with the same virus. No effect of the reovirus type 3 on the adult mice could be elicited.In contrast, the suckling mice infected by the reovirus type 3 revealed a definite change in pancreatic zymogen enzymes. However, the zymogen enzymes were affected in a nonparallel fashion and three groups of enzymes with different responses were noted. Amylase and lipase activities were significantly diminished (P < 0.001) at 6 days of viral infection. The endopeptidases, trypsin (P < 0.025) and chymotrypsin (P < 0.001) activities were increased significantly in the infected group. The exopeptidases, carboxypeptidase A and B in the infected animals were not changed significantly compared to the control.It seems reasonable that the reovirus type 3 infection in the suckling mouse causes diminished lipase and amylase activities that might contribute to the pathogenesis of viral enteritis.Speculation: Studies on viral enteritis in infants and young animals have primarily implicated changes in the small intestine as the cause of diarrhea. The viral invasion of the intestinal mucosa causes villous cell destruction and as a consequence, the mucosa generated is immature and incapable of handling normal salt and water absorption.In addition, changes in pancreatic function as a result of an extension of the viral infection to the pancreatic parenchyma might contribute to the pathophysiologic mechanisms operating in viral enteritis.In the infected suckling mice, only amylase and lipase activities are diminished to a large extent, while trypsin and chymotrypsin activities are elevated and carboxypeptidase A and B activities remain unaffected. The nonparallel change in pancreatic enzymes toward a viral insult can be explained by a separate effect of the virus on the biosynthesis of each of the zymogen pancreatic enzymes. It is conceivable that amylase and lipase while in a developing stage, are more affected by the virus than the other pancreatic enzymes which are already developed to a certain extent. Another explanation is that lipase and amylase activities are intrinsically more prone to be decreased in response to different disease states affecting the pancreas. Diminished lipolytic and amylolytic activities due to viral gastroenteritis is a possible contributing factor of the diarrhea in infants and children.

Methotrexate (MTX) effect on pancreatic enzymes in leukemic mice.

We evaluated the effects of MTX on normal and leukemic mice (L-1210) in order to determine the effects of MTX on the pancreas in both leukemic and normal control mice. The mice were divided into two experiments according to their treatment schedule. The first group consisted of mice with L-1210 leukemia and normal controls treated with escalating doses of MTX or with placebo at 10, 15, 20, 25, 30, or 45 mg/kg. These mice were all arbitrarily sacrificed at 48 hr. The second group consisted of mice with L-1210 leukemia and normal controls treated with an arbitrary dose of MTX (20 mg/kg) or with placebo and sacrificed at spaced time intervals of 12, 24, 48, and 96 hr after MTX administration. Both L-1210 and normal nonleukemic mice were examined for histological changes in the pancreas as well as pancreatic zymogen enzyme levels. In general, the maximal effect on the pancreas occurred at a dose of 20 mg/kg of MTX and at 48 hr following MTX administration. Histologically, in the L-1210 leukemic mice, there was leukemic infiltration, diminished acini, and fatty changes in the pancreas; these findings were less pronounced following treatment with MTX compared to those treated with placebo. All pancreatic enzymes in untreated leukemic mice were significantly diminished (P<0.001) compared to normal mice. Amylase and lipase were decreased more than trypsin and chymotrypsin. The zymogen enzymes in MTX-treated normal mice responded in a hyperbolar fashion, falling to a nadir at 20 mg/kg of MTX. In contrast, in the MTX-treated leukemic mice, the zymogen enzymes increased significantly reaching a peak at 20 mg/kg of MTX and then falling off with increasing MTX doses.

Letter to the editor: importance of technique in the induction of glucuronyl transferase in Gunn rats.

Letter to the Editor: Importance of Technique in the Induction of Glucuronyl Transferase in Gunn Rats