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Background and Aim: The bone-related peptide osteoprotegerin has been linked to vascular calcification and peripheral vascular disease. We investigated the association between osteoprotegerin and development of foot complications in persons with type 1 diabetes., Materials and Methods: Prospective observational study of 573 persons with type 1 diabetes, 225 women; age [mean±SD] 42.3±10.3years. Plasma osteoprotegerin was measured by ELISA., Results: Median (IQR) osteoprotegerin was 2.80(2.35-3.63)μg/L and follow-up time (median (range)) was 12.7(0.1-15.6)years. Endpoints included: new foot ulceration (n=153), Charcot foot (n=14), vascular surgery/amputation (n=53), loss of foot pulse (n=57), and peripheral neuropathy (n=99). In unadjusted analyses, higher osteoprotegerin was associated with development of all endpoints (p≤0.026). Higher osteoprotegerin remained associated with development of foot ulcer, and the combination of vascular surgery/amputation, loss of foot pulse and neuropathy (p≤0.001) in a sex and age adjusted model. After further adjustment (nephropathy status, smoking, HbA 1c , systolic blood pressure, serum cholesterol, high sensitivity C-reactive protein, eGFR, and presence of neuropathy and/or claudication and/or foot ulcer at baseline), higher osteoprotegerin remained associated with development of foot ulcer (HR (95% CI) per doubling: 1.75 (1.04-2.97); p=0.037)., Conclusion: Higher osteoprotegerin levels were associated with development of foot ulcer, even after comprehensive adjustment., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A novel approach, which is based on the analysis of sequences of images recorded using energy-filtered transmission electron microscopy and can be used to assess the reaction of a solid with a gas at elevated temperature, is illustrated for the reduction of a NiO/ceramic solid oxide fuel cell anode in 1.3mbar of H2. Three-window elemental maps and jump-ratio images of the O K edge and total inelastic mean free path images are recorded as a function of temperature and used to provide local and quantitative information about the reaction kinetics and the volume changes that result from the reaction. Under certain assumptions, the speed of progression of the reaction front in all three dimensions is obtained, thereby providing a three-dimensional understanding of the reaction., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The current understanding in the study of focused electron beam induced processing (FEBIP) is that the growth of a deposit is mainly the result of secondary electrons (SEs). This suggests that the growth rate for FEBIP is affected by the SE emission from the support. Our experiments, with membranes thinner than the SE escape depth, confirm this hypothesis. We used membranes of 1.4 and 4.3 nm amorphous carbon as supports. At the very early stage, the growth is support-dominated and the growth rate on a 4.3 nm thick membrane is three times higher than on a 1.4 nm thick membrane. This is consistent with Monte Carlo simulations for SE emission. The results suggest that SEs are dominant in the dissociation of W(CO)6 on thin membranes. The best agreement between simulations and experiment is obtained for SEs with energies between 3 and 6 eV.With this work we revisit earlier experiments, working at a precursor pressure 20 times lower than previously. Then, despite using membranes thinner than the SE escape depth, we did not see an effect on the experimental growth rate. We explain our current results by the fact that very early in the process, the growth becomes dominated by the growing deposit itself.

Objective: The soluble urokinase plasminogen activator receptor (suPAR) is a plasma marker of low grade inflammation and has been associated with cardiovascular risk. We wanted to investigate whether suPAR was associated with markers of subclinical organ damage., Methods: In a population sample of 2038 individuals, aged 41, 51, 61 and 71 years, without diabetes, prior stroke or myocardial infarction, not receiving any cardiovascular, anti-diabetic or lipid-lowering medications, we measured urine albumin/creatinine ratio (UACR), carotid atherosclerotic plaques and carotid/femoral pulse wave-velocity (PWV) together with traditional cardiovascular risk factors and high sensitivity C-reactive protein (hsCRP)., Results: suPAR was significantly associated with the presence of plaques (P = 0.003) and UACR (P < 0.001), but not PWV (P = 0.17) when adjusting for age, gender, systolic blood pressure, cholesterol, plasma glucose, waist/hip ratio, smoking and hsCRP. However, suPAR explained only a small part of the variation in the markers of subclinical organ damage (R(2) 0.02-0.04). During a median follow-up of 12.7 years (5th-95th percentile 5.1-13.4 years) a total of 174 composite endpoints (CEP) of cardiovascular death, non-fatal myocardial infarction and stroke occurred. suPAR was associated with CEP independent of plaques, PWV, UACR, and hsCRP as well as age, gender, systolic blood pressure, cholesterol, plasma glucose, waist/hip ratio and smoking with a standardized hazard ratio of 1.16 (95% confidence interval 1.04-1.28, P = 0.006)., Conclusion: suPAR was associated with subclinical organ damage, but predicted cardiovascular events independent of subclinical organ damage, traditional risk factors and hsCRP. Further studies must investigate whether suPAR plays an independent role in the pathogenesis of cardiovascular disease., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Bilirubin is toxic in most biological systems tested. Several mechanisms have been suggested for this toxic effect, including inhibition of enzyme systems and inhibition of cell regulatory reactions (protein/peptide phosphorylation). The identity of the basic mechanism(s) has not been conclusively proven, but inhibition of peptide phosphorylation, perhaps mediated or modulated by lysine at the active site(s), appears to be compatible with many of the observations currently found in the literature. Bilirubin entry into brain is facilitated by drug displacement of bilirubin from its albumin binding, reduced albumin binding capacity, increased brain bloodflow, increased permeability of the blood-brain barrier, and other factors. The rate of bilirubin entry into brain, as well as the degree of retention and rate of clearance from brain, depends on which of these circumstances are operative. It is as yet unclear whether the mechanism responsible for increased brain bilirubin is important for toxicity. The mechanism for preferential localization of bilirubin to the basal ganglia in kernicterus is also not known. Bilirubin appears to distribute differentially to brain subcellular compartments and is oxidized in brain by an enzyme localized on the inner mitochondrial membrane. This enzyme is found both in neurons and in glia, but appears to be more active in the latter. The activity increases with postnatal age, and is subject to genetic variability in animals. The enzyme is cytochrome c-dependent. It is as yet not clear whether the activity of this enzyme serves a brain-protective effect in severe hyperbilirubinemia.

Insight into the location, state, and function of a promoter in heterogeneous catalysis was obtained through atomic-resolution in situ transmission electron microscopy. In the most active ruthenium catalyst for ammonia synthesis known so far, the barium promoter is shown to be located in two different phases in the catalyst. The increased activity is suggested to be related to a two-dimensional barium-oxygen overlayer on the ruthenium crystals. The possibility for conducting such studies for other reactions could add substantially to our current understanding of heterogeneous catalysis. Heterogeneous catalysis plays an increasingly important role in environmental protection processes, in fuel upgrading, and in providing the majority of the chemical building blocks required by contemporary society. Most heterogeneous catalysts of industrial importance are multicomponent materials that are designed by trial-and-error experimentation. Application of even the most sophisticated physical-chemical characterization techniques is usually not sufficient to obtain a complete understanding of the structure of the active site, the reaction mechanism and kinetics, the structural dynamics, and the specific roles of all catalyst components.

Twenty-four studies filled the inclusion criteria for a Cochrane report on the use of fiberoptic phototherapy for neonatal jaundice. Fiberoptic phototherapy lowers serum bilirubin and may have a place in the treatment of neonatal jaundice. In term and near-term infants fiberoptic phototherapy is inferior to conventional phototherapy, whereas in premature infants the effects are comparable. A better effect is achieved when conventional and fiberoptic phototherapy are combined as compared to conventional phototherapy alone. Fiberoptic phototherapy has not been shown to interfere less with parent-infant bonding than conventional phototherapy. There continues to be a great need for research concerning phototherapy for neonatal jaundice.

Background: P-glycoprotein (Pgp) is an ATP-dependent, integral plasma-membrane efflux pump that is constitutively expressed on (i) adult apical brush-border epithelial cells of the intestine, (ii) the bile canalicular face of hepatocytes, and (iii) the brush border epithelium of renal proximal tubules. This Pgp tissue distribution and localization affects the absorption, distribution, metabolism, and excretion of Pgp substrates. Little is known regarding the ontogeny of Pgp expression in these tissues., Methods: Postnatal expression of Pgp on brush border membranes of small intestine, liver, and kidney as a function of maturity from birth through adulthood was determined using Western immunoblotting and immunohistochemical techniques. Tissue was isolated from FVB mice at four different ages: day of life 0 (D0), day of life 7 (D7), day of life 21 (D21), and adult (Ad). The relative expression of Pgp protein on Western immunoblots was assessed by scanning densitometry and indexed as a percentage (mean+/-SEM) of the adult levels., Results: On Western immunoblots, Pgp expression was limited at birth (19+/-6% of Ad) and increased significantly with maturation in intestine (ANOVA, P<0.005). In contrast, hepatic (113+/-12% of Ad) and renal (96+/-15% of Ad) Pgp expression were at adult levels at birth. The tissue-specific developmental pattern of Pgp expression was confirmed by immunohistochemistry., Conclusions: We conclude that Pgp is expressed in a tissue-specific and developmentally regulated fashion and speculate that developmental modulation of intestine-Pgp expression may affect the oral bioavailability of Pgp substrates.

Bilirubin is a product of heme catabolism which by virtue of its lipid solubility can cross the blood-brain barrier and enter the brain. Neonatal jaundice is a common transitional phenomenon which is due to the combination of increased heme catabolism and rate limitations as far as hepatic conjugation and biliary excretion of bilirubin. In the great majority of cases this is an innocuous condition, which is even posited to have some beneficial effects due to the ability of bilirubin to quench free oxygen radicals. However, because bilirubin is neurotoxic, hyperbilirubinemia in the newborn may exceptionally result in death in the neonatal period, or survival with severe neurological sequelae (kernicterus). Bilirubin enters the brain through an intact blood-brain barrier. Clearance of bilirubin from brain partly involves retro-transfer through the blood-brain barrier, and possibly also through the brain-CSF barrier into CSF. Work in our lab during the past 5 years has substantiated earlier work which had suggested that bilirubin may also be metabolized in brain. The responsible enzyme is found on the inner mitochondrial membrane, and oxidizes bilirubin at a rate of 100-300 pmol bilirubin/mg protein/minute. The enzyme activity is lower in the newborn compared with the mature animal, and is also lower in neurons compared with glia. Studies of different rat strains have documented genetic variability. The enzyme is cytochrome-c-dependent, but has as yet not been unequivocally identified. The rate of oxidation of bilirubin is such that this enzyme probably contributes meaningfully to the clearance of bilirubin from brain., (Copyright 2000 Academic Press.)

Neonatal jaundice must have been noticed by caregivers through the centuries, but the scientific description and study of this phenomenon seem to have started in the last half of the 18th century. In 1785 Jean Baptiste Thimotée Baumes was awarded a prize from the University of Paris for his work describing the clinical course in 10 jaundiced infants. The work by Jaques Hervieux, which he defended for his doctor of medicine degree in 1847, was, in many respects, a landmark. He had autopsied 44 jaundiced infants and apparently had clinical observations on many others. His descriptions of pathoanatomical findings were very detailed and systematic. A number of his clinical observations are still thought to be accurate today, such as the essentially benign nature of neonatal jaundice in most cases, the appearance of neonatal jaundice during the first 2 to 4 days of life as well as its disappearance within 1 to 2 weeks, and the cephalocaudal progression of jaundice. He described jaundice of the brain in 31 of his 44 autopsied cases, with variable intensity of staining. Johannes Orth was an assistant to the famous Virchow in Berlin, when in 1875 he published the results of an autopsy of a jaundiced term infant. The brain was notable for an intense yellow staining of the basal ganglia, the wall of the third ventricle, the hippocampus, and the central parts of the cerebellum. While the contribution of Orth was limited to this single case report, in 1903 Christian Schmorl presented the results of his autopsies of 120 jaundiced infants to the German Society for Pathology. All of these infants' brains were jaundiced, but only 6 cases demonstrated a staining phenomenon similar to that previously described by Orth. Schmorl coined the term kernicterus (jaundice of the basal ganglia) for this staining pattern. Although the following century of scientific study has added an enormous amount of information about the epidemiology and pathophysiology of neonatal jaundice and kernicterus, the contributions of Hervieux, Orth, and Schmorl will undoubtedly continue to be seen as historical landmarks in our quest for understanding of these phenomena.

Brain mitochondrial membranes oxidize bilirubin at a rate that may be biologically significant. Hyperosmolality delays clearance of bilirubin from rat brain. We hypothesized that this might be due to a decrease in brain bilirubin metabolism. Rats were anesthetized and infused with 75 mM/kg urea i.v. over 5 min (n = 7) and sacrificed at 1 h; control rats (n = 7) had no treatment. Crude mitochondrial membrane fractions ('P2') were produced by homogenization and differential centrifugation in sucrose. The change in optical density at 440 nM of a 10-micromol/l bilirubin solution was measured after 60 min incubation with mitochondrial membranes. Hyperosmolality did not affect bilirubin oxidation by brain mitochondrial membranes (t = 1.27, p = 0.23 by unpaired t-test). The lack of effect of hyperosmolality on bilirubin oxidation by brain mitochondrial membranes suggests that decreased clearance of bilirubin from brain in hyperosmolality may be related to changes in transfer of bilirubin across the blood-brain barrier., (Copyright 2000 S. Karger AG, Basel)

Native complement factors and complement activation products were measured in healthy neonates (n = 72) and in a group of infants with premature prolonged rupture of the membranes (PPROM) without sepsis (n = 10). Vitronectin concentration in normal cord blood was not correlated with gestational age, and the median value was 86.0% of adult values. This was markedly higher than other native complement factors studied (factor B: 35.9%, C4: 45.1%, C3: 56.2%). The concentration of C9 showed a positive correlation with gestational age and was very low, 10.8% of normal adult values in cord blood and 8.3% in the patients. Fifteen percent of the neonates had C9 levels lower than 2% of adult values. The complement activation products Bb and SC5 b-9 were significantly elevated in the patients (159% and 130% of control values, respectively), indicating alternative and terminal pathway activation. In contrast, C4 bc and C3 bc levels were not increased. The maximum amount of SC5 b-9 which could be generated in the neonatal sera by cobra venom factor was highly correlated with C9 concentration (rs = 0.86, p = 0.0001) The profound C9 deficiency found in neonates is correlated with gestational age, limits the capacity to form bacteriolytic C5 b-9 (m) and may predispose for severe invasive bacterial infection. The plasma level of SC5 b-9 under normal conditions was very low, only 0.3% (0.1%-3.0%) of the values obtained after CVF activation of the same samples. Therefore, we suggest that the analysis of SC5 b-9 is applicable also in neonates, in spite of their extremely low C9 levels.

Bilirubin is a well-known neurotoxin and presents a particular problem in newborn infants. This is partly due to the high incidence of unconjugated hyperbilirubinemia in that age group, but may also be due to increased vulnerability to bilirubin toxicity. The brain may be able to protect itself against bilirubin toxicity through a process of oxidation. The responsible enzyme is localized on the inner mitochondrial membrane and appears to be more active in glia than in neurons and to increase in activity with postnatal maturation. Here we have investigated the possibility that the responsible enzyme might be a cytochrome oxidase, malate dehydrogenase, or monoamine oxidase, all enzymes located on the inner mitochondrial membrane. Mitochondria were obtained from rat brains through homogenization and differential centrifugation in sucrose medium. The ability of mitochondrial membranes to oxidize bilirubin was measured by following the change in optical density at 440 nm of a bilirubin solution to which a membrane suspension had been added. The activity was not changed by in vitro inhibitors of malate dehydrogenase or monoamine oxidase, but was moderately inhibited by ketoconazole and clotrimazole, both known inhibitors of hepatic cytochrome P450 oxidases. Activity was inhibited by depletion of cytochrome c in the mitochondria and reconstituted by reintroducing cytochrome c into the reaction mixture. The reaction was not modified by the addition of a free radical quencher, but was inhibited by removal of oxygen from the reaction mixture. The activity was significantly inhibited by cyanide. Activity was retained in a 100,000-g pellet and was not influenced by the addition of NAD, NADP, NADH, NADPH, GSH, or GSSH to this pellet. We conclude that the bilirubin-oxidizing activity in brain mitochondrial membranes is cytochrome c dependent, but does not appear to be unequivocally identifiable as a cytochrome P450 oxidase., (Copyright 1999 Academic Press.)

Jaundice is a common reason for therapeutic intervention in newborn infants and phototherapy is effective treatment if enough light energy is delivered to a skin surface area of sufficient size. Narrow spectrum blue light is superior to white light, but in developing countries fluorescent blue lamps often have to be imported and are much more expensive than white lamps. We developed a phototherapy unit in which seven daylight fluorescent tubes are placed immediately under the floor of a transparent plexiglass crib. The efficacy of this unit, delivering approximately 19 microW/cm2/nm, was compared with that of two conventional phototherapy units using overhead lamps placed 35 cm above the infants. One unit used daylight fluorescent tubes and delivered approximately 4 microW/cm2/nm, the other unit used special blue fluorescent tubes and delivered approximately 22 microW/cm2/nm. Fifty-one infants were included in the analyses, all of them breastfed on demand. Serum bilirubin levels were determined spectrophotometrically at 0, 12 and 24 h. The decrement in serum bilirubin concentrations was significantly greater in infants undergoing phototherapy with the new device or with special blue lamps compared to conventional overhead daylight lamps (p < 0.001 both at 12 and at 24 h). We conclude that highly efficient phototherapy may be delivered with daylight fluorescent lamps placed in very close proximity to the patient. Thus, lack of access to expensive imported special blue lamps does not preclude delivery of effective phototherapy in developing countries.

Although the well-known neurotoxic agent bilirubin can induce alterations in neuronal signaling, direct effects on neurotransmitter release have been difficult to demonstrate. In the present study we have used permeabilized nerve terminals (synaptosomes) from rat brain prelabeled with [3H]norepinephrine to examine the effects of bilirubin on transmitter release. Rat cerebrocortical synaptosomes were permeabilized with streptolysin-O (2 U/ml) in the absence or presence of bilirubin (10 microM-320 microM) and Ca2+ (100 microM), and the amount of radiolabeled transmitter released during 5 min to the medium was analysed. Low levels of bilirubin decreased Ca2+-evoked release in a dose-dependent manner, with half-maximal effect at approx 25 microM bilirubin. Higher levels of bilirubin (100-320 microM) increased [3H]norepinephrine efflux in the absence of Ca2+, suggesting that high bilirubin levels induced leakage of transmitter from vesicles. The nontoxic precursor biliverdin had no effect on Ca2+-dependent exocytosis. Our data indicate that bilirubin directly inhibits both exocytotic release and vesicular storage of brain catecholamines.

Early discharge from maternity units has become more frequent over the last decade. The driving forces have been historical, ideological, logistic, and financial. Often early discharge has been implemented without adequate changes in follow-up practices, resulting in increased readmission rates to hospitals. Many infants recalled for metabolic screening have not showed up, with the attending risk of mental retardation. The question of length of maternity stays has not been adequately studied. Established practice should therefore be changed cautiously. Mechanisms for quality control must be in place to ensure early detection of unanticipated problems. Early discharge from neonatal intensive care units is happening in several countries, either due to lack of equipment or bed space, or to reduce the cost of care. In wealthier countries technology-dependent infants are increasingly being cared for at home. These practices have also not been well studied, and probably should be. Of particular interest is the question of parents' ability to cope with the challenges of caring for these infants.

P-glycoprotein (P-gp), encoded by the mdr1a gene, is an ATP-dependent plasma membrane protein that is expressed in abundance on the blood-brain barrier (BBB). P-gp limits the CNS influx and retention of a variety of lipophilic compounds. We hypothesized that brain bilirubin content after an i.v. bilirubin infusion would be increased in P-gp-deficient mdr1a null mutant transgenic mice (mdr1a(-/-)) compared with controls. Eighteen mdr1a(-/-) null mutant and 18 P-gp-sufficient wild type mice (+/+) were anesthetized and 50 mg/kg bilirubin infused through the tail vein. Brain bilirubin content (mean +/- SEM) 10 min after infusion was significantly higher in mdr1a(-/-) (18.1 +/- 2.4 nmol/g) compared with (+/+) mice (10.4 +/- 1.0 nmol/g). Brain bilirubin content declined 60 min after infusion but remained higher in mdr1a(-/-) (10.3 +/- 1.4 nmol/g) compared with (+/+) mice (5.3 +/- 0.9 nmol/g). Brain bilirubin clearance did not differ between groups (t 1/2 approximately 55 min). We conclude that P-gp-deficient mdr1a(-/-) mice have significantly higher brain bilirubin content compared with controls after an i.v. bilirubin load. These data suggest that 1) bilirubin is a substrate for P-gp and 2) the increased brain bilirubin content in mdr1a(-/-) mice is due to enhanced brain bilirubin influx. We speculate that BBB P-gp provides a protective effect against bilirubin neurotoxicity by reducing brain bilirubin influx.

Gap junctions are conductive channels that connect the interiors of coupled cells. We determined whether gap junctions propagate transcellular signals during metabolic stress and whether such signaling exacerbates cell injury. Although overexpression of the human proto-oncogene bcl2 in C6 glioma cells normally increased their resistance to injury, the relative resistance of bcl2+ cells to calcium overload, oxidative stress and metabolic inhibition was compromised when they formed gap junctions with more vulnerable cells. The likelihood of death was in direct proportion to the number and density of gap junctions with their less resistant neighbors. Thus, dying glia killed neighboring cells that would otherwise have escaped injury. This process of glial 'fratricide' may provide a basis for the secondary propagation of brain injury in cerebral ischemia.

Uptake of neurotransmitters into synaptic vesicles occurs through specific transport proteins which are driven by an ATPase-generated electrochemical force consisting of a proton gradient and a membrane potential. In this study we examined the effects of bilirubin, a well known neurotoxic agent, on the vesicle uptake both of [3H]dopamine (which is driven mostly by the proton gradient) and [3H]glutamate (which is driven mostly by the membrane potential), and compared these to the vesicular proton gradient, which was estimated by analyzing the uptake of [14C]methylamine. Bilirubin inhibited the uptake of both dopamine and glutamate (p < 0.01), with an identical dose-response curve for both transmitters. Inhibition was detected readily at 75 microM. The effects of bilirubin were dependent on the concentration of vesicles in the assay, suggesting that the concentration of bilirubin in the membranes and not the water phase was important. Bilirubin also decreased uptake-dependent efflux of dopamine from the vesicles. In contrast, bilirubin had no effect on the vesicular proton gradient, as measured by methylamine uptake. Our results show that bilirubin has essentially identical inhibitory effects on the uptake of both a monoamine transmitter and an amino acid transmitter into synaptic vesicles, but does not influence the vesicular H+-ATPase or proton translocation. Our data suggest an inhibitory interaction between bilirubin and several transport proteins in synaptic vesicle membranes.

Gap junctions are highly conductive channels that allow the direct transfer of intracellular messengers such as Ca2+ and inositol triphosphate (IP3) between interconnected cells. In brain, astrocytes are coupled extensively by gap junctions. We found here that gap junctions among astrocytes in acutely prepared brain slices as well as in culture remained open during ischemic conditions. Uncoupling first occurred after the terminal loss of plasma membrane integrity. Gap junctions therefore may link ischemic astrocytes in an evolving infarct with the surroundings. The free exchange of intracellular messengers between dying and potentially viable astrocytes might contribute to secondary expansion of ischemic lesions.

Neonatal haemochromatosis is a disorder which affects foetuses and newborns. It is characterized by hepatocellular insufficiency, often appearing on the first day of life in the form of coagulopathy, hypoalbuminemia, hypoglycemia and jaundice. While spontaneous recovery has been reported, most of these infants die, and the diagnosis was previously often made during autopsy. With the help of MRI and salivary gland biopsies, plus increasing awareness of this disorder, the diagnosis is now often made quite early, and successful liver transplantations have been reported. Recently, there have also been encouraging preliminary reports of successful intervention with antioxidant and chelation pharmacotherapy, using a combination of selenium, vitamin E, N-acetylcysteine, deferoxamine, and prostaglandin E. We describe two patients with neonatal haemochromatosis who were both treated with this new "cocktail", one of whom died at five days of age, while the other survived, but needed a liver transplant at 2 1/2 months of age. The pathology of this condition is characterized by hepatic cirrhosis with giant cell transformation, and by siderosis of extrahepatic tissues. The prognosis is poor, and our experience with antioxidant treatment has been disappointing. Liver transplantation is a therapeutic option, but its use is limited by the scarcity of donor organs and the small size of many of the patients.

Bilirubin is metabolized by brain mitochondrial membranes. This activity increases with postnatal age, is higher in glia than in neurons, and is subject to genetic variability. The rates of oxidation are such that this mechanism probably contributes meaningfully to clearance of bilirubin from brain. Phenobarbital is a well-known inducer of bilirubin-metabolizing enzymes in the liver. Previous incomplete data had suggested that phenobarbital treatment might also affect bilirubin metabolism in brain. We studied the clearance of bilirubin from brain (branch 1) as well as the oxidation of bilirubin by brain mitochondrial membranes (branch 2) following pretreatment of young adult SPRD rats with phenobarbital 75 mg/kg i.p. daily for 7 days; controls received solvent only. On day 8, rats in branch 1 (n = 8 in both phenobarbital-treated and control groups) were anesthetized and 50 mg/kg bilirubin was infused i.v. over 5 min. Rats were sacrificed after 60 min and brain bilirubin determined by acid chloroform extraction. In branch 2 (n = 7 in both groups) rats were sacrificed with i.p. pentobarbital, brains were homogenized in 0.32 M sucrose and mitochondria separated out by differential centrifugation. The mitochondrial membrane suspension was added to a 10-mumol/1 bilirubin solution, and the rate of bilirubin oxidation was measured at 440 nm. As expected following phenobarbital treatment, serum bilirubin was slightly lower in phenobarbital-treated vs. control rats [126 +/- 32 vs. 140 +/- 21 mumol/1 (mean +/- SD); p = 0.3]. Brain bilirubin values were also lower in the phenobarbital-treated rats (0.44 +/- 0.06 vs. 0.52 +/- 0.08 nmol/g, p = 0.044). As the lower brain bilirubin values in phenobarbital-treated rats could be due entirely to lower serum bilirubin values, we compared the brain:serum bilirubin ratios in the two groups. These were not different (0.0063 +/- 0.0016 vs. 0.0064 +/- 0011). The rate of bilirubin oxidation was significantly lower in phenobarbital-treated vs. control rats (256 +/- 10 vs. 286 +/- 26 pmol/min/mg protein, p = 0.023). We conclude that phenobarbital inhibits bilirubin metabolism in brain. However, albeit statistically significant, the difference is small and may not be biologically meaningful. Thus, pretreatment with phenobarbital had no effect on overall brain bilirubin clearance rate. We speculate that phenobarbital treatment may be neither helpful nor harmful relative to the neurotoxic effects of bilirubin in infants with already established jaundice.

Sepsis is believed to increase the risk of bilirubin brain toxicity, but the mechanism is not known. Adult male Sprague-Dawley rats were injected intraperitoneally with either 20 mg/kg Escherichia coli lipopolysaccharide, approximately 5 x 10(9)/kg CFU Listeria monocytogenes or vehicle 48 h prior to sacrifice. Rats were killed with an intraperitoneal injection of pentobarbital. Mitochondrial membrane fractions were produced by homogenization of the brains and differential centrifugation in 0.32 M sucrose. The mitochondrial pellet was resuspended in distilled water and sonicated to rupture the mitochondria. The protein concentration of the suspension was standardized to 2.5 mg/ml. Bilirubin oxidation was assayed in a pH 8.2, 0.1 M barbital buffer containing 10 microM bilirubin, 5 mM EDTA, and 500 U/ml catalase. Optical density was measured at 440 nm before and after a 60-min incubation at 37.5 degrees C. There were no differences between the control, endotoxemic, and septic groups as far as the ability of brain mitochondrial membranes to oxidize bilirubin (bilirubin oxidation rate: 289 +/- 11 vs. 295 +/- 9 vs. 296 +/- 12 pmol/min/mg protein, mean +/- SD). We conclude that endotoxemia or sepsis do not change the ability of brain mitochondrial membranes to oxidize bilirubin. If sepsis truly increases the risk of bilirubin encephalopathy in neonatal jaundice, this is likely to involve other mechanisms.

Background: Neonatal hemochromatosis (NH), also known as perinatal hemochromatosis or neonatal iron storage disease, is a disorder in fetuses and newborn infants. A retrospective study was conducted to report management of patients with NH., Methods: Retrospective analysis was conducted by chart review and by review of histologic material from patients with NH., Results: Neonatal hemochromatosis was diagnosed in 14 patients between 1985 and 1995. All were considered for orthotopic liver transplantation (OLTX). From 1993 onward, all patients were treated with an antioxidant-chelation "cocktail," consisting of deferoxamine, vitamin E, N-acetylcysteine, selenium, and prostaglandin-E1. Of 6 patients with NH diagnosed before 1993, 4 underwent OLTX; only 1 is still alive. Of 8 patients with NH diagnosed after 1993 and treated with the cocktail, 7 expired before OLTX. One stabilized on therapy, but having never recovered full synthetic liver function, underwent OLTX and is now alive and well., Conclusion: Neonatal hemochromatosis carries a grim prognosis; however, successful OLTX is curative. The use of an antioxidant-chelation cocktail did not improve outcome in the patients studied. Earlier (perinatal) diagnosis may be required for optimal results. Further study of other interventions, including antenatal diagnosis and earlier institution or modification of cocktail therapy appears warranted.

Several lines of evidence indicate that the extent of ischemic injury is not defined immediately after arterial occlusion, but that infarction expands over time. Episodes of spreading depression have been linked to this secondary increase in infarct volume. Tissue bordering the infarction fails to repolarize following spreading depression and is incorporated into the lesion. The result is that ischemic infarctions expand stepwise after each episode of spreading depression. Another line of evidence has demonstrated that gap junction blockers effectively inhibit spreading depression. These observations suggest that traffic of potentially harmful cytosolic messengers between ischemic cells and surrounding nonischemic cells might cause amplification of injury in focal stroke. It is therefore conceivable that minimizing gap junction permeability might reduce final infarct volume. To test this hypothesis, the authors pretreated rats with the gap junction blocker, octanol, before occluding the middle cerebral artery and compared the sizes of the ischemic lesions to those in rats that received the vehicle, dimethyl sulfoxide, prior to arterial occlusion. Histopathological analysis was performed 24 hours later. The 12 octanol-treated animals showed a significantly decreased mean infarction volume (80 +/- 16 mm3) compared with the nine control rats (148 +/- 9 mm3). In a separate set of experiments, the frequency of experimentally induced waves of spreading depression was evaluated after octanol treatment. Octanol pretreatment resulted in complete inhibition in two of nine animals, transient inhibition in five, and no inhibition in two. The results indicate that gap junction inhibitors, when not limited by toxicity, have significant therapeutic potential in the treatment of acute stroke.

Protein phosphorylation is an important mechanism for regulation of cell processes. Bilirubin inhibits phosphorylation of several peptides/proteins by a number of different kinases, and this may contribute to the toxic effects of bilirubin on cells, particularly neurons. Bilirubin binds to lysine residues on both albumin and ligandin. The ATP-binding subdomain II on protein kinases contains an invariant lysine, which might hypothetically be involved in mediation or modulation of bilirubin-inhibition of protein phosphorylation. We have studied the ability of lysine-containing peptides to modulate the effects of bilirubin, using phosphorylation of a phospholemman peptide catalyzed by protein kinase A as a model system. Addition of bilirubin (50 microM) decreased the activity of the catalytic subunit of protein kinase A by 75%. A synthetic lysine-containing decapeptide which mimicked part of subdomain II on the protein kinase family was partially able to prevent the bilirubin effects. Similar effects were not observed with two other decapeptides in which lysine had been replaced by arginine or alanine. Polylysine (100 microM) completely prevented the inhibitory effect of 50 microM bilirubin, whereas polyglutamate and polyarginine did not have this effect. Poly-D-lysine and poly-L-lysine appeared to be equivalent in their ability to prevent the bilirubin effect. These data support the notion that binding of bilirubin to lysine may play a role in the mediation and/or modulation of bilirubin neurotoxicity.

Bilirubin is oxidized by brain mitochondrial membranes at a rate which may contribute significantly to clearance of bilirubin from brain. Different strains of congenitally jaundiced rats (Gunn rats) vary widely as far as the mortality rate of the homozygous (jaundiced) pups. Because the ability to oxidize bilirubin in brain may protect against toxicity, we hypothesized that the ability to oxidize bilirubin would be lower in Gunn rat strains (ACI/N-j) with a high mortality rate in the homozygous pups. Mitochondria were obtained from young rat brains by differential centrifugation in sucrose gradients. The mitochondria were ruptured by sonication. The change in optical density of a bilirubin solution at 440 nm was measured over time following addition of the membrane suspension. The rate of bilirubin oxidation was significantly lower in rats of the RHA/N-j strain both at 7-8 days of age and in adults, compared to rats of the ACI/N-j and the Sprague-Dawley strains at the same age points. Differences in mortality rates between the RHA/N-j and the ACI/N-j strains of Gunn rats could not be explained on the basis of differences in the ability of brain mitochondrial membranes to oxidize bilirubin, as these activities were lower in the RHA/N-j rats, which also have lower mortality rates, but higher in the ACI/N-j rats, which have remarkably high mortality rates. This study also confirmed previous findings relative to age maturation of the enzyme activity., (Copyright 1997 Academic Press.)

Four infants below 6 months of age with proven respiratory syncytial virus infection in need of assisted mechanical ventilation were successfully treated by high-frequency oscillatory ventilation. One of the four infants fulfilled the criteria for extracorporeal membrane oxygenation before the start of oscillation, and one on the second day on high-frequency oscillatory ventilation. However, extracorporeal membrane oxygenation was not needed in any of the infants. All survived, and three appeared to be without any pulmonary sequelae.

Approximately 0.5-1% of all newborns are born infected with cytomegalovirus (CMV), but of these only one out of ten show symptoms at birth, most often with hepatosplenomegaly, thrombocytopenia, and/or brain affection. Of the remaining nine, one may later develop sequelae with hearing loss and/or mental retardation. CMV infection may also be acquired perinatally or in the newborn period, and may cause pneumonia and/or sepsis, possibly also gastrointestinal symptoms like blood in the stool, and poor weight-gain. We have diagnosed CMV infection in ten neonates and infants, and describe these patients in terms of symptoms, diagnosis and treatment. Ganciclovir is being tested in clinical trials as a treatment for congenital CMV infection, and was given to two of our patients with apparently good results.

Bilirubin is oxidized by brain mitochondrial membranes at a rate which may contribute significantly to clearance of bilirubin from the brain. Neurons appear to be more sensitive to bilirubin toxicity than glial cells. Clinical experience has suggested that sensitivity to bilirubin neurotoxicity may be greater in the neonate than later in life. We hypothesized that the ability to oxidize bilirubin would be lower in mitochondrial membranes from a pure neuronal compared to a mixed glial/neuronal source, and lower in immature than more mature brains. Mitochondria of synaptosomal and nonsynaptosomal origin were obtained from young rat brains by differential centrifugation in sucrose gradients. Synaptosomes were lysed by hypoosmotic treatment, and mitochondria were ruptured by sonication. The change in optical density of a bilirubin solution at 440 nm was measured over time following addition of the membrane suspension. The rate of bilirubin oxidation was significantly higher in nonsynaptic than in synaptic mitochondrial membranes [99.1 +/- 42.3 (mean +/- SD) vs 69.9 +/- 30.9 pmol/ min/mg protein, t = 4.835, P = 0.0003]. "Crude" mitochondrial membranes were obtained by differential centrifugation in sucrose from the forebrains of rats of 7, 14, and 21 days postnatal age as well as adults, and from rabbits of 1 and 7 days postnatal age as well as adults. In both species the rates of bilirubin oxidation increased significantly with postnatal age (rats: F = 55.3, P < 0.0001; rabbits: F = 101, P < 0.0001). Mitochondrial membranes from a pure neuronal source oxidize bilirubin at a significantly lower rate than membranes from a mixed glial/neuronal source. This suggests that neurons may be less able to detoxify bilirubin locally and thus might contribute to the apparent higher sensitivity to bilirubin toxicity in these cells vs glia. Similarly, the lower bilirubin-oxidizing ability of mitochondrial membranes from immature brains seems compatible with the clinical impression of increased vulnerability to bilirubin neurotoxicity in the newborn.

Jaundice is common in neonates and often requires diagnostic and therapeutic intervention. Recently a case has been made for less aggressive intervention when jaundice occurs in healthy term infants. This issue is also being discussed by Norwegian pediatricians. The present paper compares the most commonly used guidelines in Norwegian pediatrics with the results of a recent international survey of therapeutic practices in cases of neonatal jaundice. The survey documented wide variation in the approach to this common problem. Norwegian intervention limits for premature/SGA infants are generally at or above the 90th centile for the international sample, and must be said to be fairly unaggressive. The Norwegian intervention limits for term infants are generally between the median and the 75th centile. Thus they are on the less aggressive side of the international sample. Though it is likely that many term infants can tolerate quite high serum bilirubin levels without significant risk of brain damage, arguments for further elevation of the intervention limits are problematic as long as we do not have the tools to identify individuals, possibly very few, with lower tolerance.

The neurotoxic effects of bilirubin may involve modulation of neuronal protein phosphorylation systems. Using in vitro phosphorylation assays and a variety of protein substrates and purified protein kinases, we have studied the mechanism of bilirubin-induced inhibition of protein phosphorylation. Bilirubin was found to inhibit cAMP-dependent, cGMP-dependent, Ca(2+)- calmodulin-dependent, and Ca(2+)-phospholipid-dependent protein kinases, irrespective of substrate properties. Fifty percent inhibition occurred at bilirubin concentrations varying from 20 to 125 microM. Kinetic analysis, using the isolated catalytic subunit of cAMP-dependent kinase and a synthetic peptide substrate derived from the protein phospholemman, indicated that bilirubin (50 microM) decreased the apparent Vmax of the reaction, irrespective of whether ATP or peptide levels were varied, without significantly altering the apparent K(m) value. Thus our results indicate that bilirubin can inhibit catalytic domain(s) of protein kinases by apparent noncompetitive mechanism(s), presumably by interacting with noncatalytic domains on the enzyme. Given the key role of protein phosphorylation in cellular regulation, the widespread inhibitory effect of bilirubin on protein kinases may contribute to bilirubin neurotoxicity.

Jaundice is one of the most common clinical phenomena in the neonatal period and a frequent indication for treatment with phototherapy, exchange transfusion, or drugs. The present study documents the variability in approaches to the treatment of this condition. A mail questionnaire was sent to neonatal units worldwide. One hundred and eight answers (49% response rate) were received from Europe (n = 72), North America (n = 28), Africa (n = 7), and Asia (n = 1). The neonatal intensive care units represented by the respondents had 31 +/- 18 beds [mean +/- SD], and 638 +/- 519 admissions per year. All units offered phototherapy, 106/108 performed exchange transfusion, while 44/108 used some form of drug therapy. There was considerable variability among the units in their approaches to the jaundiced neonate. This applied to all aspects of care, including type of phototherapy lights used, practical implementation of phototherapy, use of fluid supplementation, and use of prophylactic phototherapy. The majority used written protocols for investigation and treatment of neonatal jaundice and would let their decision on whether to treat be influenced by the infant's clinical state. There was great variability between units in the level of serum bilirubin that would trigger therapy. This applied across weight groups and to phototherapy as well as exchange transfusion. The significant heterogeneity in our approach to the treatment of jaundiced neonates suggests that our understanding of the biology of neonatal jaundice is inadequate and that further research will be necessary in order to provide a more solid biological foundation for therapy.

Bilirubin may be cleared from the brain by transport across the blood-brain barrier and by a 'sink effect' into the cerebrospinal fluid. However, there is also evidence to suggest that bilirubin may be metabolized in the brain by a process of oxidation. The purpose of this study was to confirm the existence of bilirubin metabolism in the brain and to examine the possible contribution of such an activity to the bilirubin staining pattern characteristic of kernicterus. Mitochondrial membrane fractions were prepared in 0.32 M sucrose from whole rat brains as well as brain regions. The change in optical density of a bilirubin solution at 440 nm was measured over time following addition of the mitochondrial suspension. Our results confirmed the existence of a bilirubin-metabolizing activity in brain mitochondrial membranes. This activity could be removed by heating the mitochondrial suspension and had a definable temperature and pH maxima. The rate of oxidation of bilirubin ranged from 109 to 164 pmol/min/mg protein. There were significant differences between rat brain regions in the ability to oxidize bilirubin. However, these differences could not explain the kernicterus staining phenomenon, because the highest activities were found in brain regions which are more heavily stained in kernicterus. We conclude that bilirubin is metabolized by brain mitochondrial membranes at a rate that would appear to represent a biologically meaningful contribution to bilirubin clearance from the brain. In the present model, differences in such an activity between brain regions do not appear to be compatible with a kernicteric staining pattern.

The risk of bilirubin encephalopathy is believed to be increased in hemolytic relative to nonhemolytic jaundice. Young SPRD rats were injected with either acetylphenylhydrazine (APHZ, n = 22) 75 mg/kg or an equivalent volume of the solvent (control, n = 22) intraperitoneally for 3 successive days. One the 4th day, hyperbilirubinemia was induced by a 5-min intravenous infusion of 50 mg/kg bilirubin. After sacrifice (at 10 or 60 min) the brain vessels were flushed in situ with ice-cold saline. Serum bilirubin was 600 +/- 108 mumol/l (mean +/- SD) at 10 min, and 295 +/- 98 mumol/l at 60 min. Hematocrit was significantly reduced in the APHZ rats versus controls (26 +/- 3 vs. 41 +/- 3, p < 0.0001). The concentration of bilirubin in brain was determined by acid chloroform extraction and diazotization. The brain bilirubin values were 5.5 +/- 1.5 versus 5.4 +/- 2.1 mmol/g at 10 min (APHZ vs. control), and 1.3 +/- 0.6 versus 0.8 +/- 0.7 nmol/g at 60 min. The half-life of bilirubin in brain was calculated with an exponential fitting program. The half-life of bilirubin in brain for the two groups was 24.3 +/- 21.9 versus 18.5 +/- 28.3 min. There were no significant differences between the groups in either of these measures. We conclude that in young rats, hemolytic anemia does not increase the acute entry of bilirubin into brain, nor does it affect the clearance of bilirubin from brain.

In spite of adequate dietary regimen, many patients with galactosemia have developmental abnormalities. We studied 8 patients with galactosemia, aged 9 months to 19 years who had all been treated with a galactose-free diet from an early stage. Neurological functioning, general developmental, and language and speech development were assessed in all cases. The results show that even medically well treated children and young adults with galactosemia are at risk to develop disabilities, including mental retardation, speech and language disabilities. Verbal dyspraxia was diagnosed in 3 of 6 patients, who had acquired verbal language, all with IQ 70 or below. This may indicate that verbal dyspraxia is just one symptom among others in patients with galactosemia and mental retardation.

Brain bilirubin concentrations are increased by hyperosmolality and hypercarbia, but the mechanism is not known. The same applies to the mechanism for preferential localization of bilirubin to basal ganglia. Young Sprague-Dawley rats were used. Groups were: control (n = 15), hypercarbia (n = 16, pH approximately 6.95), and hyperosmolality (n = 13, serum osmolality approximately 390 mosm/L). Hyperbilirubinemia was induced by a 5-min infusion of 50 mg/kg bilirubin, containing approximately 20 microCi [3H]bilirubin. Rats were killed at 15-min intervals up to 60 min, and the brains were flushed in situ, dissected into seven regions, weighed, and dissolved. Brain bilirubin was determined by scintillation counting. The half-life of bilirubin in brain was calculated by exponential fitting, which also allowed an estimation of brain bilirubin at the end of the bilirubin bolus. The kinetics of bilirubin clearance from brain were first order. The half-life of bilirubin in brain was significantly prolonged in hyperosmolality (38.2 +/- 28.8 min [mean +/- SD]) compared with control (16.1 +/- 7.7 min) and hypercarbia (12.6 +/- 8.6 min) (F = 12.6, p < 0.0001 after log transformation) results. The estimated acute entry of bilirubin into brain was significantly increased in hypercarbia (13.9 +/- 7.4 nmol/g) compared with control (5.6 +/- 2.1 nmol/g) and hyperosmolality (6.5 +/- 2.1 nmol/g) (F = 19.2, p < 0.0001 after log transformation) results. There were no significant differences between brain regions in acute entry or clearance of bilirubin. The kinetics of increased brain bilirubin differ between hypercarbia (increased acute entry) and hyperosmolality (delayed clearance). Preferential localization of bilirubin to basal ganglia is not produced under, and may not be explained by, the conditions investigated.

Oxygenation problems are common in very sick infants, and involve a high risk of mortality or residual morbidity. We describe two infants with severe oxygenation difficulties. In one baby these were caused by meconium aspiration, while in the second, premature (1,500 g birth weight) infant Escherichia coli septicemia had precipitated persistent pulmonary hypertension. Inhaled nitric oxide led to immediate improvement in oxygenation, and the one infant was weaned off mechanical ventilatory support after four days, the other after ten days. There were no discernible side effects in either baby. Nitric oxide treatment should be considered for infants with life-threatening oxygenation problems who do not respond adequately to conventional management.

Ethical dilemmas are a daily occurrence in neonatal intensive care units. Some of these questions relate to life-and-death issues such as whom to treat or not to treat, if and when to discontinue life support, and organ donation. However, other issues are just as important in daily practice. These include the choice of a framework for ethical discussions, the question of who participates in the discussions, the parents' right to participation and information, the process and timing of decision-making, the ethics of clinical research in subjects unable to consent, respect and communication between health professionals, and the limits of confidentiality. In most of these dilemmas there is no quick and easy route to the one and only true answer. Neonatal health care workers are frequently confronted with ethical dilemmas, and are forced to reflect ethically. In this process, it is essential to be able to listen attentively to others, health workers as well as lay persons, and to integrate their opinions into one's own reflections. These two factors together must form the basis for daily clinical work and for ethical decisions.