In vitro metabolism of putrescine by diamine oxidase in tissues of the pregnant rat

The metabolism of¹⁴C-putrescine by diamine oxidase was tested on the 19th day of pregnancy in different organs of rats. The highest turnover was found in maternal placenta and uterus where 94% of the reaction product was identified as ¹-pyrroline. Considerable deamination rates were also observed in ovary, liver and fetal placenta where, however, -aminobutyric acid was the predominant product. In the kidney only a small diamine oxidase activity was measured. No formation of spermidine or spermine from¹⁴C-putrescine was detected.     

Aspects on diamine oxidase activity and its determination.

Homogenate of guinea-pig liver and human placenta, tissues known to be rich in diamine oxidase, were incubated with 14C-putrescine and the metabolites formed were determined. In the incubate of guinea-pig liver, the major metabolites were GABA and some unidentified compound(s); delta1-pyrroline and 14CO2 were also obtained. In both the maternal and fetal parts of human placenta, radioactive GABA and the unidentified compound(s) as well as delta1-pyrroline were found. The results indicate that GABA is an important intermediate in putrescine metabolism. Determination of the amount of delta1-pyrroline is thus not well suited as a measure of the diamine oxidase activity in tissues.     

Electron microscopy of the initial stages of placentation in the pig

Summary To elucidate the morphology of the initial stages of epitheliochorial placentation in the pig, material from 10 sows of the Danish Landrace and from one Göttinger minipig gilt from day 13 to day 26 of gestation was processed for scanning and transmission electron microscopy. The observed foetomaternal interaction from day 19 1/2 minipig placenta corresponded well to the observations on the Danish Landrace placenta. From the results and the discussion it was concluded that the following structures were implicated in the initial phases of placentation in the pig: (1) Protruding epithelial proliferations of the uterine epithelium enclosed by chorionic caps serving to immobilize the blastocyst (days 13 and 14). (2) A thick glycocalyx on the maternal and a thin one on the foetal epithelium before contact. (3) Close apposition between the apical plasma membranes from trophoblastic and uterine epithelium (day 14). (4) Development of interdigitating microvilli (days 15–16). (5) Formation of apical domes on the uterine epithelium closely related to the trophoblast provided with long cytoplasmic extensions into a luminal space between the apical domes, apparently representing a transition from histiotropic to haemotrophic nutrition (days 15–20). (6) Placentation, development of interdigitating microvilli between foetal and maternal epithelium, was extended but not terminated in the peripheral zone at day 26.     

Determination of histaminase (diamine oxidase) activity byo-dianisidine test: Interference of ceruloplasmin

Until nowo-dianisidine was used as an indicator substance in a test system for the determination of diamine oxidase. More recently, however, this substance was also used to measure ceruloplasmin activity. A study of the test principles revealed thato-dianisidine was the one denominator for both enzymes. As it was found for diamine oxidase the indicator was oxidized via peroxidase mediated H₂O₂ cleavage. Ceruloplasmin, however, oxidizedo-dianisidine directly with resulting free radical formation.An addition of histamine dihydrochloride or putrescine dihydrochloride to an incubation mixture, containing ceruloplasmin as enzyme ando-dianisidine orp-phenylene-diamine as substrates, produced an activation of the enzyme, being more than 10-fold in the presence of 1×10^–2 M putrescine at pH 7.0. It was assumed that an allosteric effect of the dihydrochloride component might be responsible for this activation.When the activity of purified diamine oxidase was determined by theo-dianisidine test and by the isotope assay, a very good correlation between both methods was found. But, in a mixture of diamine oxidase and ceruloplasmin, no differentiation between the two enzymic activities by theo-dianisidine test was possible. This observation demonstrated an interference of ceruloplasmin when theo-dianisidine method was used for the determination of diamine oxidase activity.To apply our findings also in vivo the amine oxidase activity increasing in guinea-pig plasma during inflammation, was determined by theo-dianisidine test and by specific methods for some amine oxidases. Despite an enhanced oxidation of theo-dianisidine observed, only an increase of ceruloplasmin activity was found. It was concluded that ceruloplasmin had no histaminase activity as has been assumed by other authors using theo-dianisidine test.     

Aspects on diamine oxidase activity and its determination

Homogenate of guinea-pig liver and human placenta, tissues known to be rich in diamine oxidase, were incubated with ¹⁴C-putrescine and the metabolites formed were determined. In the incubate of guinea-pig liver, the major metabolites were GABA and some unidentified compound(s); Δ¹-pyrroline and ¹⁴CO₂ were also obtained. In both the maternal and fetal parts of human placenta, radioactive GABA and the unidentified compound(s) as well as Δ¹-pyrroline were found. The results indicate that GABA is an important intermediate in putrescine metabolism. Determination of the amount of Δ¹-pyrroline is thus not well suited as a measure of the diamine oxidase activity in tissues.     

Transamination of L-cysteine sulfinate in the growing rat

The enzyme activities involved in the transamination of L-cysteine sulfinate (L-alanine 3-sulfinic acid), L-aspartate and L-cysteine were examined in fetal, neonatal and maternal rat liver and placenta. In fetal and neonatal rat liver, aminotransferase activity was most active with L-cysteine sulfinate as a substrate and was also active with L-aspartate, while activity with L-cysteine was very low. The activity of transamination of L-cysteine sulfinate in rat liver developed in parallel with that of L-aspartate and L-cysteine. The aminotransferase activity markedly increased after the 19th day of gestation, reaching the same value as adult liver on the 3rd day after birth. The ratios of transamination of L-cysteine sulfinate to that of L-aspartate and to that of L-cysteine were constant during development. These observations suggest that L-cysteine sulfinate, L-aspartate and L-cysteine are transaminated by the same enzyme in the rat liver during development. Since placental aminotransferase activity was extremely low compared with that of the liver, it was suggested that the placenta did not play an important role in the transamination of these amino acids during pregnancy.     

Uptake of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and the N-methyl-4-phenylpyridinium ion (MPP+) into fetal mouse brain through the placenta

The neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was found to be taken up into fetal mice through the placenta from a maternal mouse. C57 black mice were mated and MPTP (30 mg/kg) was given intramuscularly at the 18th day of gestation. Definite amounts of MPTP were detected in fetal brains by assay using high-performance liquid chromatography (HPLC) at 1 h after MPTP injection, and much higher contents of MPTP were found in maternal brains, too. An oxidative product of MPTP, the N-methyl-4-phenylpyridinium ion (MPP+), was also detected in brains of both mother and fetus and its concentrations in their brains were followed at 1, 3, 6, 12, 24 h after MPTP injection. The time to reach the maximal MPP+ concentration in brains was different between mother and fetus; 1 h and 3 h respectively. In addition to brain, considerable amounts of MPP+ were found in fetal liver, maternal liver and kidney, and in the placenta.     

Diamine oxidase activity and related substrates in rat liver after chronic ethanol feeding

Chronic ethanol feeding as 12% or 36% of total calories caused a dose-dependent diminution of diamine oxidase activity in rat liver. Hepatic cadaverine and histamine levels were unmodified by ethanol, whereas putrescine increased, partially in relation to the decrease in diamine oxidase activity. Such results may be of interest in view of an aggravation of ethanol-induced hepatic damage when exogenous diamines and polyamines reach the liver in potentially toxic amounts.     

Taurine concentrations in fetal, neonatal and pregnant rats

The concentrations of taurine in the fetal and neonatal organs, and the maternal organs, plasma and urine of rats between the 15th day of gestation and the 21st day after birth were determined using an automatic amino acid analyzer. In the fetal liver and brain and in the placenta, the taurine concentration was the highest of all ninhydrin positive compounds. In the fetal liver and placenta, the concentrations of taurine increased significantly with the gestational days. Concentrations of taurine in the brain were much higher in the fetus and neonate than that in the adult. Moreover, the total amount of taurine per fetus increased markedly after the 15th day of gestation, and near term, reached almost the same amount as in the adult rat liver. In contrast to this, a significant decrease was observed in the taurine concentration in the maternal liver and muscle near term. The concentration of taurine in the urine of pregnant rats decreased near term, but in the plasma of pregnant rats the concentration of taurine did not change during pregnancy.     

Effects of in vivo and in vitro exposure to rhodamine dyes on mitochondrial function of mouse embryos

Cationic rhodamines (Rh 123 and Rh 6G) can cause developmental toxicity in mice and inhibit embryonic mitochondrial respiration following in vivo or in vitro dye exposure. Rh B, a neutral rhodamine, fails to show such effects at comparable doses. To assess effects of rhodamines on development, F0F1ATPase activity and ADP translocation were measured on gestation day (GD) 12 in embryonic and adult mitochondria. ATP synthesis in embryonic mitochondria transplacentally exposed to Rh 123 (15 mg/kg/day) or Rh 6G (0.5 mg/kg/day) given to dams by i.p. injection from GD 7 to 10 were inhibited 39% and 49%, respectively. When isolated mitochondria were treated, dose-dependent inhibition was seen; at 5 micrograms of dye/mg mitochondrial protein, ATP synthesis was inhibited 65% and 81% by Rh 123 and Rh 6G, respectively. When F0F1ATPase activity was assessed, in vitro Rh 123 and Rh 6G exposures at levels up to 8 micrograms/mg mitochondrial protein resulted in enzyme inhibition, but at 10 micrograms/mg, ATPase activity was stimulated. Uncoupler-stimulated ATPase activity was also inhibited. ADP translocation was decreased by 19.1% and 37.7% by Rh 123 and Rh 6G, respectively, at dye concentrations of 20 micrograms/mg. Results of in vitro exposure of maternal liver mitochondria were similar to those for embryonic mitochondria, whereas liver from dams exposed in vivo on GD 7-10 was unaffected on GD 12. In vivo or in vitro treatment with Rh B did not affect any embryonic or maternal parameters. Such results support the hypothesis that inhibition of mitochondrial energy metabolism is a mechanism for the developmental toxicity of cationic rhodamines.     

Effect of selected immunoregulatory agents on low-grade contact sensitivity

The effect of selected compounds with known immunoregulatory activity was examined in a 45-h sensitization period oxazolone contact-sensitivity reaction. Oxazolone sensitivity was induced by applying 0.1 ml of 5% oxazolone in absolute ethanol to the shaved abdomen of C57B1/6 mice on day 0. Challenge with oxazolone followed 45 h later and was accomplished by painting a 5% solution of oxazolone in absolute ethanol on the left hindpaw. The response at 24 h was determined plethysmographically. Histamine (0.062–1.0 mg/kg, subcutaneously, twice a day), concanavalin A (0.31–5.0 mg/kg, intravenously), penicillamine (6.25–25 mg/kg, subcutaneously), chloroquine (6.25– 25 mg/kg, subcutaneously), and thymosin fraction 5 (0.125–1.25 mg/kg subcutaneously) all stimulated the oxazolone reaction when administered on day 0. These data suggest that the low-grade oxazolone response may be a useful assay to detect immunostimulatory activity.     

Diamine oxidase activity and imidazoleacetic acid formation in the foetal and maternal guinea pig liver

Diamine oxidase activity and imidazoleacetic acid formation in the foetal and maternal guinea pig liver during gestation were examined. DAO activity and IMAA formation in the foetal liver increased continuously, while maternal enzyme activity and ImAA formation in the second half of pregnancy simultaneously decreased. The roles of GABA and ImAA are discussed.     

Specific temperature dependence of diamine oxidase activity and its thermal stability in the presence of Polyvinylalcohol

An inflexion point on the dependence of the swine kidney diamine oxidase activity upon the temperature was found at 40–43°C, suggesting a conformational transition. The activation energies with putrescine as substrate calculated from the Arrhenius plots were 38.23 kcal/mol for the temperature interval 25–40°C and only 15.14 kcal/mol for the range 45–60°C. These values suggest two different conformations, one corresponding to the interval below 40°C and another one between 43–60°C, with an intermediate transitory form corresponding to the inflexion point at 40–43°C. For various temperature decades within 10–60°C, peculiarQ ₁₀ values in the range 1.37–3.00 (differing from the usual valueQ ₁₀=2), were obtained. The non-strictly Arrhenius curves, the activation energies and the inflexion point were quite similar with and without 0.05% polyvinylalcohol. This particular temperature effect found for swine kidney diamine oxidase is similar to the one reported for bovine serum amine oxidase. An increased enzyme thermal stability was obtained in the presence of high molecular weight polyvinylalcohol.     

Diamine oxidase in the hen

In adult hens diamine oxidase (histaminase) activity was found in gastrointestinal tract (with the highest value in ileum), liver and spleen. Intestinal diamine oxidase is predominantly a particle-bound enzyme. In the intestine oxidation of putrescine leads to Δ-pyrroline formation, in liver both Δ¹-pyrroline and γ-aminobutyric acid are formed. The inhibitor properties of hen intestinal and rat intestinal diamine oxidases are very similar and differ from pea seedling diamine oxidase. The natural dipeptides carnosine and anserine are relatively potent inhibitors of hen intestinal diamine oxidase.     

Immunohistochemical localization of histaminase (diamine oxidase) in decidual cells of human placenta.

An immunohistochemical technique has been developed for the localization of histaminase (diamine oxidase) in human placenta. The procedure utilizes antibody to highly purified human placental enzyme prepared in rabbits. Histaminase is shown to be present in the cytoplasm of the decidual cells of the maternal placenta; the staining is specific and can be removed by preabsorption of the antisera with purified antigen. The present demonstration of histaminase in the maternal portion of placenta confirms previous biochemical data suggesting that the enzyme is not a fetal product in this tissue. This study also provides further evidence that the presence of histaminase in some human tumors and in normal tissues like kidney and intestine is the result of the expression of a mature and not a fetal genome.     

Location and activities of acetylcholinesterase and butyrylcholinesterase in the rat and human placenta

The location and physiological functions of acetylcholinesterase and butyrylcholinesterase in the placenta are still debated. In the present study the activities of both enzymes were studied histochemically in the rat and human placenta, using an optimized Karnovsky/ Roots method. Additionally, they were measured biochemically. Acetylcholinesterase was active in the syncytiotrophoblast, cytotrophoplast cells and the visceral and parietal yolk sac epithelial cells of the rat (n = 10) and in the syncytiotrophoblast, cytotrophoblast cells, endothelial cells and the media of fetal blood vessels of the human placenta (n = 9). Butyrylcholinesterase could not be detected histochemically. Biochemically measured levels at certain developmental stages of the placenta revealed maximum acetylcholinesterase activity in the 8th week p.m. human placentae (102.9 nmol·min^–1 per mg protein), 35% lower activity in the 12th week p.m., and minimum (44.1 nmol-min^–1 per mg protein) in term placentae. In contrast, maximum butyrylcholinesterase activity was measured in week 12 p.m. (106.9 nmol·min^–1 per mg protein). In rat placentae, butyrylcholinesterase activity on gestational day 21 reached 150% of the level on gestational day 16. Acetylcholinesterase activity remained constant. In placentae of pre-eclamptic patients, acetylcholinesterase and butyrylcholinesterase activities were found to be increased by 16% and 45%, respectively. The results suggest that placental acetylcholinesterase can no longer be considered as derived from maternal blood, but is primarily located within rat and human placental tissue.Supported by grants P-8737-MED and P-9140-MED from the Austrian Science Foundation (FWF).A preliminary account of this work was presented at the 9th International Congress of Histochemistry and Cytochemistry, Maastricht, Netherlands, 08/30-09/05, 1992     

Human intestinal diamine oxidase (DAO) activity in Crohn's disease: A new marker for disease assessment?

The key-enzyme for the metabolism of diamines in man is diamine oxidase (DAO). Its highest activities are in the intestinal mucosa, localized in the cytoplasm of the mature enterocytes of the small and large bowel. If the gut is affected by inflammation in Crohn's disease macroscopical changes are observed. This prospective study investigated if these mucosal alterations are also reflected in changes of mucosal diamine oxidase activity and/or mucosal histamine content respectively. Twenty patients (12 female, 8 male; age: , range 18 49 years) undergoing gut resection because of complications in Crohn's disease (Jan.–Dec. 1988) formed the basis of the study. Tissue samples of the resected material from areas inflamed and histologically not involved in the disease were investigated for diamine oxidase activities and histamine content. Diamine oxidase activities in the mucosa obtained from the macroscopically normal proximal (155.6; (76–393) mU/g ( range)) and distal (132; (58.5–295) mU/g) resection margins were similar to our previous findings. In all patients, however, samples from the diseased mucosa had significantly (ca. 50%) lower diamine oxidase activities (74.5; (5–262) mU/g) compared to the healthy tissue. Similar differences were found in material obtained either from whole intestinal wall or from the mucosa. The determination of diamine oxidase activity constitutes possibly a more unambiguous and earlier parameter for assessing the extent of the inflamed area than histological disease presentations. Using biopsies the necessary extent of resection could be estimatedbefore operation: this may influence operative strategies and help in the definition of the minimum amount of inflamed gut to be removed.Supported by grant of Deutsche Forschungsgemeinschaft (Lo 199/15-2).     

Guanabenz as inhibitor of copper-containing amine oxidases

The effects of guanabenz on some copper containing amine oxidases are described. Guanabenz in vitro inhibits pig plasma benzylamine oxidase with a IC₅₀ M 5.1±0.8×10^–6 M. It also inhibits pig kidney diamine oxidase and rat liver mitochondrial monoamine oxidase at higher concentrations. The significance of this property of guanabenz is discussed.     

Effect of feeding on the diurnal rhythm of plasma cortisol and adrenocorticotrophic hormone concentrations in the pregnant ewe and sheep fetus.

The effects of two different feeding regimes on the 24 h profiles of maternal and fetal plasma cortisol and adrenocorticotrophic hormone (ACTH) concentrations were studied in eight pregnant ewes between 123 and 144 days of gestation. Once daily-fed ewes (n = 4) received 1 kg of lucerne-chaff at 11.00 h, and multi-fed ewes (n = 4) received 100-200 g of lucerne-chaff at 09.00, 11.00 and 13.00 h and then 150 g until 09.00 h the following day. There were significant differences between the two feeding groups in the 24 h profile of maternal plasma osmolality; once daily feeding at 11.00 h was associated with a peak in maternal plasma osmolality at 15.00 h whereas maternal plasma osmolality reached plateau levels at around 17.00 h in the multi-fed group. There were also differences between the two feeding groups in the 24 h profiles of maternal and fetal plasma glucose. Maternal and fetal plasma glucose reached peak concentrations at 19.00 h in the once daily-fed ewes in contrast to the multi-fed group, where a plateau in maternal and fetal plasma glucose was reached between 19.00 h and 09.00 h the following day. A significant diurnal variation in the plasma concentrations of cortisol was present in the once daily-fed ewes from 123 days gestation and in their fetuses after, but not before, 135 days gestation. Plasma cortisol peaked at 11.00 h in the ewes and at 13.00 h in the fetuses of this group. In the once daily-fed group there was also a significant diurnal variation in maternal and fetal plasma ACTH; plasma ACTH concentrations were highest at 11.00 h in the ewes aged between 123 and 144 days and in fetuses after 135 days gestation. In the multi-fed group, whilst ACTH was highest at 09.00 h in the ewes and at 13.00 h in the fetuses, there was no significant diurnal variation in the plasma concentrations of cortisol in the ewes or fetuses of this group at any stage between 123 and 144 days gestation.     

Effects of acute maternal hyperglycaemia and hyperosmolality on maternofetal transfer of calcium and magnesium across the in situ perfused rat placenta.

The effects of acute maternal hyperglycaemia and hyperosmolality on maternofetal placental transfer of Ca, Mg and 51Cr-EDTA were investigated in the rat. On day 21 of gestation (term = 23 d) the fetal circulation of the in situ placenta of anaesthetised rats was perfused with a Mg-free Krebs Ringer solution and the unidirectional maternofetal fluxes of Ca (CaJmf) and Mg (MgJmf), and the unidirectional maternofetal clearance of 51Cr-EDTA ((EDTA)Kmf) were determined before and during maternal hyperglycaemia, hyperosmolality and volume expansion, attained by infusing 30% D-glucose, 25% mannitol and 0.9% saline solutions, respectively, into the maternal circulation. MgJmf was significantly reduced during glucose infusion (23.9 +/- 1.2 v 28.2 +/- 1.4 nmol min(-1) g(-1) placenta during control perfusion (mean +/- SEM); p < 0.005) and during mannitol infusion (28.2 +/- 1.0 v 33.5 +/- 1.5 nmol min(-1) g(-1) placenta; p < 0.001). CaJmf and (EDTA)Kmf were not significantly altered by maternal hyperglycaemia or hyperosmolality. There was no significant change in MgJmf during infusion of saline into the maternal circulation. Maternal plasma Na concentration was significantly reduced in both glucose and mannitol infusion experiments, whereas maternal plasma Mg concentration was significantly reduced only during glucose infusion. We postulate that the reduced maternal plasma Na concentration in the glucose and mannitol experiments might decrease MgJmf via alteration of placental Na+/Mg2+ exchange activity.