Risto Lapatto

Melatonin reduces inflammation and cell death in white matter in the mid-gestation fetal sheep following umbilical cord occlusion

The premature infant is at increased risk of cerebral white matter injury. Melatonin is neuroprotective in adult models of focal cerebral ischemia and attenuates ibotenate-induced white matter cysts in neonatal mice. Clinically, melatonin has been used to treat sleep disorders in children without major side effects. The aim of this study was to investigate the protective and anti-inflammatory effects of melatonin in the immature brain following intrauterine asphyxia. Fetal sheep at 90 d of gestation were subjected to umbilical cord occlusion. Melatonin (20 mg/kg, n = 9) or vehicle (n = 10) was administered IV to the fetus, starting 10 min after the start of reperfusion and continued for 6 h. Melatonin treatment resulted in a slower recovery of fetal blood pressure following umbilical cord occlusion, but without changes in fetal heart rate, acid base status or mortality. The production of 8-isoprostanes following umbilical cord occlusion was attenuated and there was a reduction in the number of activated microglia cells and TUNEL-positive cells in melatonin treated fetuses, suggesting a protective effect of melatonin. In conclusion, this study shows that melatonin attenuates cell death in the fetal brain in association with a reduced inflammatory response in the blood and the brain following intrauterine asphyxia in mid-gestation fetal sheep.

Endothelial Dysfunction and Xanthine Oxidoreductase Activity in Rats With Human Renin and Angiotensinogen Genes

We examined whether xanthine oxidoreductase (XOR), a hypoxia-inducible enzyme capable of generating reactive oxygen species, is involved in the onset of angiotensin (Ang) II–induced vascular dysfunction in double-transgenic rats (dTGR) harboring human renin and human angiotensinogen genes. In 7-week-old hypertensive dTGR, the endothelium-mediated relaxation of noradrenaline (NA)-precontracted renal arterial rings to acetylcholine (ACh) in vitro was markedly impaired compared with Sprague Dawley rats. Preincubation with superoxide dismutase (SOD) improved the endothelium-dependent vascular relaxation, indicating that in dTGR, endothelial dysfunction is associated with increased superoxide formation. Preincubation with the XOR inhibitor oxypurinol also improved endothelium-dependent vascular relaxation. The endothelium-independent relaxation to sodium nitroprusside was similar in both strains. In dTGR, serum 8-isoprostaglandin F2&agr;, a vasoconstrictor and antinatriuretic arachidonic acid metabolite produced by oxidative stress, was increased by 100%, and the activity of XOR in the kidney was increased by 40%. Urinary nitrate plus nitrite (NOx) excretion, a marker of total body NO generation, was decreased by 85%. Contractile responses of renal arteries to Ang II, endothelin-1 (ET-1), and NA were decreased in dTGR, suggesting hypertension-associated generalized changes in the vascular function rather than a receptor-specific desensitization. Valsartan (30 mg/kg PO for 3 weeks) normalized blood pressure, endothelial dysfunction, and the contractile responses to ET-1 and NA. Valsartan also normalized serum 8-isoprostaglandin F2&agr; levels, renal XOR activity, and, to a degree, NOx excretion. Thus, overproduction of Ang II in dTGR induces pronounced endothelial dysfunction, whereas the sensitivity of vascular smooth muscle cells to nitric oxide is unaltered. Ang II–induced endothelial dysfunction is associated with increased oxidative stress and vascular xanthine oxidase activity.

N-acetylcysteine reduces lipopolysaccharide-sensitized hypoxic-ischemic brain injury.

Maternal inflammation/infection alone or in combination with birth asphyxia increases the risk for perinatal brain injury. Free radicals are implicated as major mediators of inflammation and hypoxia‐ischemia (HI)–induced perinatal brain injury. This study evaluated the neuroprotective efficacy of a scavenging agent, N‐acetylcysteine (NAC), in a clinically relevant model.

Human cystathionine gamma-lyase: developmental and in vitro expression of two isoforms.

Cystathionine gamma-lyase (CGL) is the last enzyme of the trans-sulphuration pathway, which converts methionine into cysteine. To study the possible differences in enzymic activity of the two human cystathionine gamma-lyase isoforms characterized earlier, these were separately expressed in human kidney embryonic 293T cells. Furthermore, developmental changes in the expression of the two mRNA forms as well as the enzymic activity in human liver were studied, as it has been postulated that a change in the relative expression of CGL isoforms causes the postnatal increase in CGL activity. Transfection with the longer isoform increased the CGL activity 1.5-fold, while the activity of the cells transfected with the shorter form did not differ from the basal activity. In human liver samples, CGL activity was only detected in adult tissue (68+/-9 nmol of cysteine/h per mg of protein), whereas activity in fetal, premature and full-term neonatal liver tissue was undetectable. In contrast, strong mRNA expression of both mRNA isoforms was detected from the 19th gestational week onwards and the longer form of CGL appeared to be predominant. The expression of the two mRNA forms varied in parallel. In conclusion, we have shown that only cells overexpressing the longer form of CGL have increased activity, and CGL appears to be regulated at the post-transcriptional level during development.

Plasma 8-isoprostane is increased in preterm infants who develop bronchopulmonary dysplasia or periventricular leukomalacia

Our aim was to assess the plasma free 8-epi–prostaglandin F2α (8-isoprostane) and ascorbyl radical as risk indicators for oxidative damage in extremely low birth weight infants (ELB-WIs) and the effect of N-acetylcysteine (NAC) on these markers. Plasma samples were collected on days 3 and 7 of life from infants who were enrolled in a randomized, controlled trial in which i.v. NAC or placebo was administered to ELBWIs during the first week of life, with the aim of preventing bronchopulmonary dysplasia (BPD). Plasma 8-isoprostane was analyzed in 83 infants using an enzyme immunoassay kit. Ascorbyl radical concentration was measured in 61 infants with electron spin resonance spectroscopy. The 8-isoprostane concentrations were similar in the NAC and placebo groups. In infants who later developed BPD or died (n = 29), the median (range) 8-isoprostane concentration was significantly higher (p = 0.001) on day 3 and day 7 [50.0 pg/mL (19–360) and 57.0 pg/mL (14–460), respectively] than in survivors without BPD [n = 54; 34.5 pg/mL (5–240) and 39.5 pg/mL (7–400), respectively]. The 8-isoprostane levels increased significantly more (p < 0.05) in infants who later developed periventricular leukomalacia. NAC treatment or the later development of BPD was not related to the ascorbyl radical levels. The ascorbyl radical level decreased significantly in all groups from day 3 to day 7, but the difference between the groups was not significant. The mean (SD) ascorbyl radical level on day 3 was significantly higher (p < 0.01) in infants who later developed periventricular leukomalacia [287 (124) versus 194 (90)]. These data suggest that plasma 8-isoprostane could serve as a marker in assessing the risk for BPD development in ELBWIs.

Circulating xanthine oxidase and neutrophil activation during human liver transplantation.

BACKGROUND & AIMS Oxygen free radicals, generated by xanthine oxidase (XO) and activated leukocytes, are involved in reperfusion injury in experimental liver transplantation. The roles of XO and neutrophil activation during reperfusion in clinical liver transplantation were studied. METHODS In 10 patients undergoing liver transplantation, we assessed plasma concentrations of circulating XO by enzyme-linked immunosorbent assay (ELISA), the purine metabolites hypoxanthine, xanthine, and urate by high-performance liquid chromatography, lactoferrin by ELISA, and malondialdehyde fluorometrically up to 48 hours postoperatively. RESULTS During reperfusion after portal vein declamping, elevated plasma concentrations of XO (52.1 ng/mL [range, 8.0-440.1]), hypoxanthine (81.62 micromol/L [48.2-108.7]), xanthine (21.01 micromol/L [8.7-22.3]), and lactoferrin (532.6 ng/mL [370.4-1326.6]) were observed compared with the preoperative levels (0 ng/mL [0-12], 1.88 micromol/L [0.62-3.15], 0.95 micromol/L [0-0.41], and 164.3 ng/mL [73.7-334.1], respectively; all P < 0.05). No changes occurred in urate or malondialdehyde. After portal vein declamping, XO, hypoxanthine, and xanthine levels were substantially greater in the hepatic than portal vein (all P < 0.05). Marginal transhepatic differences occurred in lactoferrin. CONCLUSIONS Reperfusion during liver transplantation is associated with liberation of xanthine oxidase, hypoxanthine, and xanthine from the liver into the circulation. During reperfusion, intravascular neutrophil activation takes place in the hepatic circulation.

Expanding screening for rare metabolic disease in the newborn: An analysis of costs, effect and ethical consequences for decision‐making in Finland

AIM Currently, the only metabolic disorder that newborns are screened for in Finland is congenital hypothyroidism. A proposal to start a pilot study on screening for other rare metabolic diseases using tandem mass spectrometry prompted a health technology assessment project on the effect and costs of expanded newborn screening programme options. METHOD A modelling study using data from current published studies, healthcare registers and expert opinion. RESULTS The annual running cost of screening 56,000 newborns for the chosen five disorders (congenital adrenal hyperplasia, medium-chain acyl-CoA dehydrogenase deficiency [MCADD], long chain 3-hydroxyacyl-CoA dehydrogenase deficiency [LCHADD], phenylketonuria [PKU] and glutaric aciduria type 1 [GA 1]) was estimated to be euros 2.5 million or euros 45 per newborn when starting costs were included. The costs per quality-adjusted life year (QALY) gained are a maximum of euros 25,500. Prevention of severe handicap in one newborn would reduce the costs to a maximum of euros 18,000 per QALY gained. CONCLUSIONS Expanding the Finnish neonatal screening programme would require a new organization. The cost-effectiveness, resources, ethics and equity need to be considered when deciding in favour of or against starting a new screening programme.

Increased sensitivity of homozygous Sod2 mutant mice to oxygen toxicity

Induction or overexpression of pulmonary manganese superoxide dismutase (MnSOD) has been shown to protect against oxygen (O2) toxicity. Genetic inactivation of MnSOD (Sod2) results in multiple organ failure and early neonatal death. However, lungs or O2-tolerance of Sod2 knockout mice have not been investigated. We evaluated survival, lung histopathology, and other pulmonary antioxidants (glutathione cycle) of homozygous (-/-) and heterozygous (+/-) Sod2 mutant mice compared with wild-type controls (Sod2+/+) following 48 h exposure to either room air or to O2. The ability of antioxidant N-acetylcysteine to compensate for the loss of MnSOD was explored. Mortality of Sod2-/- mice increased from 0% in room air to 18 and 83% in 50 and 80% O2, respectively. N-acetylcysteine did not alter mortality of Sod2-/- mice. Histopathological analysis revealed abnormalities in saccules of Sod2-/- mice exposed either to room air or to 50% O2 suggestive of delayed postnatal lung development. In 50% O2, activities of glutamate-cysteine ligase (GCL) (previously known as gamma-glutamylcysteine synthetase, gamma-GCS) and glutathione peroxidase increased in Sod2-/- (35 and 70%, respectively) and Sod2+/- (12 and 70%, respectively) mice, but glutathione levels remained unaltered. We conclude that MnSOD is required for normal O2 tolerance and that in the absence of MnSOD there is a compensatory increase in pulmonary glutathione-dependent antioxidant defense in hyperoxia.

Xanthine Oxidoreductase Gene Expression and Enzyme Activity in Developing Human Tissues

Xanthine oxidoreductase (XOR) has been implicated in tissue injury following ischemia-reperfusion because of its ability to generate reactive oxygen species under these conditions. In order to elucidate its role in various organs, we quantified the levels of XOR mRNA expression and activity in developing human tissues. XOR gene expression was highest in the intestine and in the liver, which also showed the highest enzyme activities. By a sensitive RNA-PCR assay, low levels of the transcript were detected in the kidney, lung, cardiac muscle, and brain of all subjects studied. XOR activities followed the mRNA distribution, being low or undetectable in tissues other than the liver and the intestine.

Expression of γ-Glutamylcysteine Synthetase During Development

Prematurity has been associated with low glutathione (GSH) concentrations in bronchoalveolar lavage fluid as well as in leukocytes from tracheal aspirates and peripheral blood. To elucidate whether this is caused by deficient GSH synthesis, the expression and activity of γ-glutamylcysteine synthetase (glutamate-cysteine ligase, GCS, EC 6.3.2.2), the rate-limiting enzyme for GSH synthesis, were measured from fetal, neonatal, and adult human liver, lung, and kidney samples. The highest activity was measured in the liver, in which mRNA expression of the catalytic GCS heavy and the regulatory light subunits, as well as activity, were, on average, similar in the various stages of development. Although GCS light subunit mRNA concentrations in the lung were higher in neonates than in fetuses and adults, enzyme activities were similar. In the adult kidney, mean enzyme activity was somewhat higher than in fetal or neonatal kidney, but this may be accounted for by the variation in the small number of samples. In conclusion, GCS is expressed and active already in the second trimester and thus low GSH concentrations found in preterm neonates appear not to be explained by deficient GSH synthesis. Other factors, such as limited availability of the GSH precursor cysteine or increased GSH consumption, may account for the lower concentrations of GSH found in preterm infants.