Coding defect and a TATA box mutation at the bilirubin UDP-glucuronosyltransferase gene cause Crigler-Najjar type I disease.

Mutations at the bilirubin UDP-glucuronosyltransferase (transferase) gene in a severely hyperbilirubinemic Crigler-Najjar (CN) type I individual was compared with that in a moderately hyperbilirubinemic CN II individual. The CN-I (CF) patient in this study sustained a TATA box insertional mutation which was paired with a coding defect at the second allele, unlike all coding defects previously seen in CN-I patients. The sequence of the mutant TATA box, [A(TA)8A], also seen in the CN-II patient, was compared with that at the wild-type box, [A(TA)7A]. Transcriptional activity with [A(TA)8A] was 10-15% that with the wild-type box when present in the -1.7 kb upstream regulatory region (URR) of the bilirubin transferase UGT1A1 gene which was fused to the chloramphenicol acetyl transferase reporter gene, pCAT 1.7H, and transfected into HepG2 cells. Also, a construct with a TA deletion, [A(TA)6A], was prepared and used as a control; transcriptional activity was 65% normal. The coding region defect, R336W, seen in CF (CN-I) was placed in the bilirubin transferase UGT1A1 [HUG-Br1] cDNA, and its corresponding protein was designated UGT1A1*32. The UGT1A1*32 protein supported 0-10% normal bilirubin glucuronidation when expressed in COS-1 cells. The I294T coding defect seen at the second allele in SM (CN-II) generated the UGT1A1*33 mutant protein which supported 40-55% normal activity with a normal Km (2.5 microM) for bilirubin. The hyperbilirubinemia seen in SM decreased in response to phenobarbital treatment, unlike that seen in CF. Parents of the patients were carriers of the respective mutations uncovered in the offspring. The TATA box mutation paired with a deleterious missense mutation is, therefore, completely repressive in the CN-I patient, and is responsible for a lethal genotype/phenotype; but when homozygous, i.e. paired with itself, as previously reported in the literature, it is far less repressive and generates the mild Gilbert's phenotype.

Neonatal bilirubin production-conjugation imbalance: effect of glucose-6-phosphate dehydrogenase deficiency and borderline prematurity.

OBJECTIVE: To evaluate relations between production and conjugation of bilirubin in the pathophysiology of jaundice in glucose-6-phosophate dehydrogenase (G6PD) deficient neonates. METHODS: Term and borderline premature (35-37 weeks gestational age), healthy, male, G6PD deficient neonates were studied close to the beginning of the 3rd day. Blood carboxyhaemogobin corrected for inspired CO (COHbc; an index of bilirubin production) and serum total conjugated bilirubin (TCB; a reflection of bilirubin conjugation) were measured in simultaneously drawn blood samples by gas chromatography and reverse phase high performance liquid chromatography respectively. A bilirubin production-conjugation index comprising COHbc/TCB was determined; a high index reflects imbalance between the bilirubin production and conjugation processes. COHbc and TCB individually and the production-conjugation index were studied in relation to serum total bilirubin (STB) concentration. RESULTS: Fifty one G6PD deficient neonates were sampled at 51 (8) hours. COHbc values did not correlate with STB (r=0.22, p=0.15). TCB did correlate inversely with STB (r=-0.42, p=0.004), and there was a positive correlation between the production-conjugation index and STB (r=0.45, p=0.002). The production-conjugation index (median (interquartile range)) was higher in the premature (n=8) than term neonates (2.31 (2.12-3.08) v 1.05 (0.53-1.81), p=0.003). This difference was the result of changes in TCB. CONCLUSIONS: The data show that jaundice in G6PD deficient neonates is the result of an imbalance between production and conjugation of bilirubin with a tendency for inefficient bilirubin conjugation over increased haemolysis in its pathogenesis. Borderline premature infants are at special risk of bilirubin production-conjugation imbalance.

Effect of various phototherapy regimens on bilirubin decrement.

Continuous phototherapy in full-term newborns was found to be more effective than intermittent illumination. Treatment efficacy was also related to age and the initial bilirubin level of the infants. In fact, the reported data indicate an increased therapeutic effect in newborns affected with nonhemolytic hyperbilirubinemia who had an initial bilirubin level greater than 15 mg/dl as compared to neonates with an initial bilirubin level less than 15 mg/dl. The light treatment was also more effective in infants older than 3 days, possibly because of an increased ligandin and conjugating capacity. Shielding the hepatic area during illumination significantly decreases the efficiency of this treatment, suggesting that the liver could also be a phototherapeutic action site.

Serum and bile bilirubin pigments in the differential diagnosis of Crigler-Najjar disease.

OBJECTIVE: To differentiate between Crigler-Najjar (CN) disease types 1 and 2. DESIGN: The patterns of serum bilirubins, bile pigment composition, and phenobarbital response were studied. PATIENTS: Three infants, affected by high serum unconjugated bilirubin concentrations, previously classified as type 1 CN. METHODS: Serum and bile bilirubin pigment composition, both before and after phenobarbital (PB) treatment, were determined by alkaline methanolysis and high-pressure liquid chromatography. PB was given for at least 3 weeks by oral administration (5 mg/kg bw per day). RESULTS: No diconjugated bilirubin was found either before or after PB treatment in the serum of the three studied infants. In two patients traces of monoconjugated bilirubin were detected before PB therapy, and the ratio of conjugated/total bilirubin (percent) was increased by the PB response. In the third patient, traces of monoconjugated bilirubin appeared only after PB administration. However, the serum unconjugated bilirubin concentration decreased significantly only in the second patient, following the second cycle of PB treatment, leading to the diagnosis of type 2 CN. The analysis of the methyl ester derivatives of bile pigments was also performed on bile samples obtained in two patients by Entero-Test (R) both before and after PB treatment. An absolute increment in monoesterified bilirubin concentration was found after PB administration, although the percent concentration increased in one case and decreased in the other. No diesterified bilirubin was detected in the bile samples. CONCLUSIONS: The present results show that in types 1 and 2 CN disease it is possible to detect traces of monoconjugated but not diconjugated bilirubin both in serum and in bile. Whereas PB treatment is effective in slightly increasing the serum monoconjugated bilirubin concentration even in type 1 CN disease, the diagnosis of type 1 or 2 is based on finding a substantial decrease of serum unconjugated bilirubin following PB administration.

Current drug treatment options in neonatal hyperbilirubinaemia and the prevention of kernicterus.

Neonatal jaundice is a frequent problem in neonatology, but the advent of phototherapy which has simplified its treatment, it no longer represents a major concern. Early hospital discharge of neonates has now resulted in a re-emergence of kernicterus. Neonatal jaundice is principally the result of a transient deficiency of bilirubin conjugation, of a partial deficiency of hepatic bilirubin uptake and intracellular transport and of an increased enterohepatic circulation of the pigment. The fact that bilirubin production in the neonate is 2 or more times greater than in the adult per kilogram of bodyweight represents the mainstay of this condition. Prevention of kernicterus in full term infants is based on the detection of neonates at risk for developing hyperbilirubinaemia, and can be accomplished with simple tests performed on umbilical cord blood such as blood type, Rh, Coombs' test and glucose-6-phosphate dehydrogenase, in order to detect haemolytic diseases. The daily evaluation of transcutaneous bilirubin measurement gives additional information on the rise of serum bilirubin level, and can help to distinguish physiological from nonphysiological hyperbilirubinaemia. A significant hyperbilirubinaemia is more frequent in infants born before term, and in neonates who do not feed well and lose more than 10% of bodyweight. In preterm infants the typical clinical feature of kernicterus is seen very rarely, and kernicterus is now a very infrequent postmortem observation. Since it is very difficult to distinguish the effects of bilirubin from other potentially toxic factors, it is difficult to give guidelines for the treatment of jaundice in very low birthweight infants other than to keep the serum bilirubin levels to a lower level than in full term infant (e.g. 10 mg/dl lower than in full term babies). The intramuscular administration of a single dose of Sn-mesoporphyrin (6 mumol/kg bodyweight) in healthy term or near-term infants seems to be a promising treatment modality for controlling hyperbilirubinaemia.

Management of neonatal hyperbilirubinaemia and prevention of kernicterus.

Hyperbilirubinaemia remains one of the most common and more important pathological conditions in the newborn. The possibility that the so-called physiological or developmental hyperbilirubinaemia, with relatively low levels of serum bilirubin, could be responsible for bilirubin encephalopathy in the small premature infant is of great concern to the neonatologist; premature newborns are prone to developing hyperbilirubinaemia. Current methodologies for suppressing severe neonatal jaundice include: (a) attempts to stimulate liver conjugating enzymes using drugs such as phenobarbital; (b) attempts to degrade bilirubin with phototherapy; and (c) exchange transfusion. It is too soon to consider tin-protoporphyrin as a drug for the prevention and treatment of neonatal hyperbilirubinaemia. However, if it can be shown that tin-protoporphyrin can serve as a safe and less costly alternate treatment, a considerable improvement in the management of neonatal jaundice would be achieved.

Postnatal development of adipose tissue in normal children on strictly controlled calorie intake.

The relations between adipose tissue development at birth, later expansion of fat mass and the behavior of fat mass and fat cell growth from birth to 12 mo of age have been studied in normal children born to normal, obese or diabetic mothers and maintained on strictly controlled calorie intake. A simple method for fat cell size determination on microsamples of fat tissue, specifically designed for small children, is reported. In the first 3 mo of life, a marked increase of fatty tissue from 13.4 +/- 0.4 to 20.3 +/- 0.8 percent of total body mass was observed. Subsequently, a sharp decrease in the relative amount of fat mass occurs, probably related to an increased energy expenditure or to a slightly higher protein content in the diet. No sex related differences in body weight, body fat mass, sum of skinfold thickness or fat cell weight were found throughout the study. No significant differences in body fat mass, sum of skinfold thickness and body fat mass as percent of body weight was observed at birth and at 3 or 6 mo of age in children of obese or gestational diabetic mothers, in comparison with children of normal mothers, and no significant correlation was found between maternal adiposity (sum of skinfold thickness or pre-gravidic overweight) or glucose tolerance (blood glucose area after OGTT) and adipose tissue development in the first 6 mo of life. Thus, in children on strictly controlled intake, obesity or diabetes in the mother do not relate to the rate of fat accumulation. Moreover, no relations were found between adipose tissue development at birth and subsequent rate of fat enlargement in the first year of life. Thus, when the interference of a different calorie intake is excluded, adiposity at birth has no predictive value for possible fatness later in infancy.

Effect of infused hypothalamic phospholipids on the maturation of foetal rabbit lungs.

The effect of hypothalamic phospholipids on the maturation of foetal lung was investigated in a series of experiments in rabbits. Sixteen pregnant does were injected with hypothalamic phospholipids (2 mg day-1 kg-1) from day 21 of gestation. A second group of 16 does was injected with saline, and served as controls. The foetuses were delivered at gestational ages between 27 and 28 days by means of caesarean section under intravenous pentothal anaesthesia. Foetuses born from treated mothers were more active, breathed better, and their lungs expanded to a greater extent compared with the foetuses born to control mothers. In the animals of treated mothers there was, on electron microscopy, evidence of accelerated maturation of alveolar epithelial cells.

Perfluorocarbons attenuate oxidative lung damage.

The aim of this study was to investigate the effect of tidal liquid ventilation (TLV) compared to conventional mechanical ventilation (CMV) on oxidative lung damage in the setting of acute respiratory distress syndrome (ARDS). After repeated lung lavages, 10 minipigs were treated with CMV or TLV for 4 hr before the animals were sacrificed. Samples for blood gas analysis and bronchial aspirate samples were withdrawn before the induction of lung injury, and at 10 min, 2 hr, and 4 hr after the beginning of ventilatory support. To assess lung oxidative damage, total hydroperoxide (TH) and advanced oxidation protein product (AOPP) concentrations were measured in bronchial aspirate samples. After 2 and 4 hr of ventilatory support, partial oxygen tension (PaO(2)) and base excess (BE) were significantly higher in the TLV group than in the CMV group, while PaCO(2) was slightly higher, but with no statistical significance. In the CMV group, the AOPP level was significantly higher at 4 hr than at baseline. TH and AOPP bronchial aspirate concentrations were higher in the CMV group than in the TLV group at 2 and 4 hr of ventilation. We conclude that animals treated with TLV showed lower oxidative lung damage compared to animals treated with CMV.

B lymphocytes in newborns.

B lymphocyte markers in 24 full term newborn infants were evaluated within four days after birth. It was demonstrated that the percentage of cells with surface immunoglobulins (SmIg) in the blood of these subjects was normal when the lymphocytes were stained with polyvalent as well as monospecific antisera. The percentage of cells bearing Fc receptors were found to be slightly, but not significantly, reduced. The ability of newborn lymphocytes to form rosettes with mouse erythrocytes was significantly reduced. The hypothesis that the B dependent system reaches complete maturity already at the first days after birth is discussed.

Plasma bilirubin level and oxidative stress in preterm infants.

OBJECTIVE: To assess the hypothesis that changes in plasma total bilirubin levels (Btot) can influence the antioxidant system and oxidative stress in preterm infants. METHODS: Twenty two healthy preterm infants who presented with visible non-haemolytic hyperbilirubinaemia were studied at the mean (SD) age of 3.7 (1.5) days. Btot, plasma total hydroperoxide concentration (TH), plasma protein SH group concentration, and total antioxidant capacity of the plasma (TAC) were measured at study entry and after 24 hours. RESULTS: Btot did not correlate with TH, TAC, or protein SH group concentration, but a significant correlation was found between TH and TAC, TH and protein SH groups, and TAC and protein SH groups, both at study entry and after 24 hours. CONCLUSION: The decrease in plasma bilirubin was contemporary with an increase in plasma antioxidant capacity and decrease in oxidative stress in preterm infants. This may be the result of the pro-oxidant effect of haem oxygenase, mediated by iron release, which may outcompete the antioxidant properties of bilirubin.

Effect of blood transfusions on oxidative stress in preterm infants.

OBJECTIVE: To confirm the increase in non-transferrin bound iron (NTBI) after packed red cell (PRC) transfusion and to evaluate the association with increased oxidative stress in preterm infants. METHOD: Twenty healthy preterm infants (gestational age 28.2 (2.2) weeks; birth weight 1047 (230) g), who required blood transfusion for anaemia of prematurity were prospectively studied. Serum concentrations of NTBI, total hydroperoxides (TH), and protein SH groups, and plasma total radical trapping antioxidant capability (TAC) were measured within three hours before and after PRC transfusion. The infants were transfused with 38.6 (23) ml PRCs over 5.8 (1.0) hours, at a mean age of 43.3 (25.1) days. RESULTS: After PRC transfusion, haemoglobin concentration increased from 9.2 (1.1) to 14.6 (1.5) g/l. Mean plasma NTBI concentration after transfusion was significantly higher (0.43 (0.45) v 2.03 (1.31) micromol/l; p = 0.001), while plasma concentrations of TH (212.3 (42.2) v 214.7 (66.3) Carr units/l) and protein SH groups (317.5 (38.8) v 353.8 (57.4) micromol/), and TAC (256.3 (36.1) v 267.1 (42.4) micromol HClO/ml) remained unchanged. CONCLUSION: For three hours after PRC transfusion, plasma NTBI is significantly increased in preterm infants, but this is not associated with significant changes in oxidative stress.

The role of blood transfusions and iron intake on retinopathy of prematurity.

BACKGROUND: The role of blood transfusions and iron intake in the pathogenesis or retinopathy of prematurity (ROP) is controversial. AIM: To evaluate the influence of packed red cell (PRC) transfusions and iron intake on ROP incidence. STUDY DESIGN: Prospective observational study. SUBJECTS: Forty-five preterm infants with birthweight <1250 g were studied. After ophthalmological study, they were divided into group A (n=24) that included newborns without ROP, and group B (n=21) that included newborns with ROP. RESULTS: Logistic regression analysis demonstrated that gestational age (OR 0.61; 95% C.I. 0.41-0.90), transfusion volume during the first week (OR 1.16; 95% C.I. 1.03-1.3) and during the first 2 months of life (OR 2.93; 95% C.I. 1.52-5.62), and iron intake during the first week of life (OR 1.15; C.I. 1.01-1.32) and during the first 2 months of life (OR 2.93; 95% C.I. 1.52-5.62) were associated with the development of ROP. CONCLUSION: Our study showed that gestational age, blood transfusion volume and iron load by transfusions are associated with the risk of occurrence of ROP in infants with a birthweight of less than 1250 g.

The effect of a 'containing' position in a hammock versus the supine position on the cutaneous oxygen level in premature and term babies.

We evaluated the influence of positioning, and particularly a containing position in a hammock compared with the supine position, on the respiration of the newborn. Recordings of the transcutaneous oxygen level of 40 preterm babies and 10 full-term babies placed in their incubators alternately in the two positions have been analyzed. We found no significant differences between the means of the oxymetric indexes in the hammock and in the supine position, even when the babies had different gestational ages and birth weights. The advantages of the containing position for the neuromotor and relational development of the newborn in the first year of life in comparison with the effects of supine placement, which should be avoided as much as possible in the premature, are pointed out.

Photochemical and photosensitizing properties of monomeric and dimeric Sn(IV)-protoporphyrin.

Sn(IV)-protoporphyrin IX (Sn-Pp) in aqueous media exists as a mixture of monomeric and dimeric species, which can be readily distinguished on the basis of their absorption maxima at around 410 and 386 nm respectively. Sn-Pp dimers prevail as the pH is decreased and are characterized by a lower fluorescence quantum yield, a larger tendency to undergo photobleaching and a reduced photosensitizing efficiency compared with the Sn-Pp monomer. The photosensitizing action of Sn-Pp appears to involve the intermediacy of singlet oxygen (1O2) as shown by photo-oxidation studies with N-acetyl-tryptophanamide in light and deuterated water solutions. Using 1,3-diphenyl-isobenzofuran as a substrate, the quantum yield of 1O2 generation by monomeric Sn-Pp was found to be about 0.6.

Pharmacokinetics of oxatomide in preterm infants.

The pharmacokinetics and tolerability of oxatomide oral suspension were investigated in preterm infants to evaluate the feasibility of planning a further study to assess its antiinflammatory effects and its effectiveness in preventing chronic lung disease (CLD). Following the administration of oxatomide 1 mg/kg, the peak plasma concentration (Cmax), the elimination half-life (t1/2), the volume of distribution (Vd), and the area under the curve (AUC) 0-36 h were measured and the following results were obtained: 42.2 +/- 15 ng/ml at 2 h after oxatomide administration, 41.4 +/- 2.0 h, 37.4 +/- 4.2 l/kg, and 468 +/- 52 ng/ml/h, respectively. Our study, therefore, demonstrated that a dose of 1 mg/kg/day oxatomide was effective in reaching therapeutic plasma levels in preterm infants without inducing adverse effects.

Efficacy and safety of the "integral" phototherapy for neonatal hyperbilirubinemia. Results of a follow-up at 6 years of age.

One hundred and ten full-term newborns were treated with integral phototherapy (IP) in the first week of life for hyperbilirubinemia (peak bilirubin concentration, 19.5 +/- 2.8 mg/dl). IP was provided by an apparatus which irradiated the infants over the entire skin surface with four visible blue light lamps placed around the body at a mean distance of only 20 cm. The irradiance of the lamps at the skin surface was 0.350 mW/sq cm, in the wavelength range between 425 and 475 nm. The IP resulted in a 48-hour bilirubin decline rate of 0.163 mg/dl/h. After a mean exposure of 78 +/- 32 h, the mean plasma bilirubin level was 8.4 +/- 0.8 mg/dl. One hundred and ten comparable nonjaundiced infants were studied as controls. At 6 years of age, both groups of subjects were called for a follow-up concerning growth, visual, and hearing functions, and neuro-developmental status. The follow-up was completed in 81 children of the IP group (73.6%) and in 89 of the controls (80.6%). There were no significant differences in the studied parameters between the two groups. The study concludes that IP appears to be an effective and safe treatment for jaundiced infants. IP employs less radiant energy from the lamp source than the traditional apparatus, but delivers this energy to a larger skin surface area.

Role of ultrasound evaluation of nuchal translucency in prenatal diagnosis.

BACKGROUND: Nuchal translucency (NT) is the ultrasonographic pattern of the accumulation of subcutaneous fluid (> or = 3 mm) behind the fetal neck. The measurement of NT thickness by ultrasound examination at 11-14 weeks of gestation has been associated with maternal age and to be an effective screening tool for trisomy 21; with an invasive method rate of 5%, about 75% of trisomical pregnancies can be identified. With the association of some biochemical markers like maternal serum free beta-human chorionic gonadotropin (beta-hCG) and pregnancy-associated plasma protein-A (PAPP-A) to ultrasonography at 11-14 weeks, it is possible to identify about 90% of chromosomal abnormalities. An increase of NT also allows us to identify most other chromosomal abnormalities, a large number of major cardiac defects, skeletal dysplasias, and genetic syndromes. In monochorial twins the discordance in the measurement of NT represents an early sign of twin-to-twin transfusion syndrome (TTTS). METHODS: The objective of our study was to assess the detection of fetal structural defects with an ultrasound scan at 11-14 weeks of gestation. We submitted 3,157 pregnant women to a routine ultrasound examination at 11-14 weeks. The patients were then submitted to ultrasound scan in the second or third trimester of pregnancy. An isolated increased NT thickness was not considered an abnormality, but these patients, nonetheless, were submitted to an early echocardiographic evaluation. Fetal structural abnormalities were classified as major or minor and of early or late onset. RESULTS: A detection rate of 4.3% (135 cases) of abnormalities was found and 22.6% of these (30 cases) were diagnosed by ultrasound scan at 11-14 weeks, including seven cardiac defects associated with increased NT. The antenatal ultrasound detection rate was 73.5%, and 33.2% were diagnosed in the first trimester assessment. A rate of 76.8% of the major defects were diagnosed by the prenatal scan and 35.8% by the scan at 11-14 weeks. Fetal structural defects with the ultrasonography at 11-14 weeks were diagnosed in about 24.3% of the cases, therefore, a second trimester abnormality is important in routine antenatal care to increase the prenatal assessment of fetal anomalies. CONCLUSIONS: As for the introduction of every new technique in routine clinical practice, the operators who perform the ultrasound scan at 11-14 weeks should be submitted to adequate training and to strict quality control.

Paralytic ileus in a mechanically ventilated preterm infant treated with fentanyl.

The administration of fentanyl for sedation of ventilated newborns can induce several side-effects such as hypertension, respiratory muscle rigidity and, as shown in this report, decreased gastrointestinal motility. We report a case of paralytic ileus in a ventilated preterm infant who was given fentanyl in the first 24 hours of life. To our knowledge, the association of paralytic ileus with fentanyl has not been reported previously in full-term or preterm infants. This study indicates that early recognition is required to shorten the delay in diagnosis.