Prenatal contact with inhalant allergens

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.     

Determination of cholesterol absorption in man by intestinal perfusion

Gel filtration was used to measure drug interaction with protein-bound bilirubin in 0.5 ml samples of Gunn rat serum, human serum and fraction V human serum albumin. Using sulfadimethoxine as a prototype differences in displacement were found in all 3 sera. The differences between human and rat serum were related to the binding characteristics of sulfadimethoxine whereas the differences between human serum and fraction V human serum albumin were attributed to displacement of bilirubin from albumin to other proteins in serum. Gel filtration permitted the use of small samples with bilirubin-albumin ratios less than 1.0 and provided data that were used for analysis of drug displacement of bilirubin using principles of drug-receptor theory. Ten of 14 drugs found to alter serum bilirubin concentrations in icteric Gunn rats had measurable effects on protein binding of bilirubin.     

A morphological and functional evaluation of transplanted isolated encapsulated hepatocytes following long-term transplantation in Gunn rats

In this study we investigated the fate of microencapsulated hepatocytes following long-term (6 months) transplantation in Gunn rats. Isolated hepatocytes were microencapsulated with a collagen matrix within an alginate-poly L-lysine composite membrane. Isolated, encapsulated hepatocytes (IEH) or free (unencapsulated) isolated hepatocytes were intraperitoneally transplanted into homozygous Gunn rats that exhibit congenital hyperbilirubinemia. Control Gunn rats received empty microcapsules. Total serum bilirubin was measured at weekly intervals for one month post-IEH transplantation, every two weeks for the next month, and monthly thereafter for up to six months. IEH samples were biopsied from the Gunn rats at monthly intervals and analyzed by light and electron microscopy. A significant (p < 0.01) decrease in total serum bilirubin was observed in IEH transplanted animals during the first month of transplantation. Thereafter, total serum bilirubin levels gradually returned to pre-transplantation levels. A mild, transient decrease in total serum bilirubin was seen in animals transplanted with free (unencapsulated) hepatocytes. No decrease in total serum bilirubin levels was seen in the Gunn rats transplanted with control (empty) microcapsules. Transplanted IEH retained its normal ultrastructure for up to one month and intact microcapsules showed no evidence of hepatocyte rejection, at this time. Degenerative changes observed in the IEH beginning at 2 months post-transplantation, suggests that repeated transplantations may be necessary for long-term effectiveness of IEH therapy.     

Development of cerebellar hypoplasia in jaundiced Gunn rats: a quantitative light microscopic analysis

The homozygous (ii) Gunn rat provides a model for hyperbilirubinemia which includes prominent cerebellar hypoplasia. Development of the Gunn rat cerebellum was examined with and without the additional effects of elevating brain bilirubin concentration to still higher levels via sulfadimethoxine (sulfa) administration. Homozygous (jj) Gunn rats and heterozygous (Nj) littermate controls (n = 32 each) were given 100 mg/kg sulfa or saline at postnatal days 3, 7, 17, and 30, and most were sacrificed 24 h later (n = 4 for each genotype at each age). Cerebellar volume, total volume and cell number for each deep cerebellar nucleus, densities for Purkinje and granule cells in the cerebellar cortex of lobules II, VI and IX, and the density of vacuolated Purkinje cells were all measured quantitatively. Cytoplasmic vacuolation provided an indication of bilirubin toxicity and was never observed in the Nj control rats. Vacuolated Purkinje cells were first observed in jj-saline rats at 18 days and were found only in the more anterior lobules of the cerebellum (II and VI). By contrast, vacuolated Purkinje cells were observed in jj-sulfa rats at both 4 and 8 days, but only in the most posterior cerebellar lobule (IX). In all older jj rats, the decline in vacuolation was accompanied by significant necrosis and resorption of the Purkinje cells in the anterior lobules. Since the Purkinje cells in the posterior lobules are the first to differentiate in the cerebellum and are resistant to bilirubin toxicity in jj-saline rats, the results support the presence of a critical period when elevated brain bilirubin may be most toxic to neuronal development. The findings suggest that neurons undergoing differentiation at the time of bilirubin exposure are most susceptible to cell death, while cells that are slightly more or slightly less mature may show only transient changes.     

Comparison of the biliary excretion of the four isomers of bilirubin-IX in Wistar and homozygous Gunn rats

The biliary excretion of the four isomers of bilirubin-IX was studied in Wistar rats (JJ) and homozygous Gunn rats (jj). Synthetic preparations of 14C-labelled pigments were used. 1. After intravenous administration, the alpha-isomer was rapidly excreted in conjugated form in bile of Wistar rats. In Gunn rats excretion was insignificant. In contrast, both rat species promptly excreted the non-alpha-isomers at rates that were comparable with that found for bilirubin-IXalpha in Wistar rats. 2. In normal rats about 16% of the beta- and delta-isomers and at least 50% of the gamma-isomer were excreted as ester conjugates of the injected parent bile pigments. Conjugation of the beta- and delta-isomers had occurred exclusively at the carboxyl groups of pyrrole ring D and C respectively. For bilirubin-IXgamma no preference for any carboxyl group could be established. 3. In homozygous Gunn rats the non-alpha-isomers were apparently excreted chemically unaltered. This suggests that, as for bilirubin-IXalpha, conjugation of the non-alpha-isomers is also deficient in Gunn rats.     

Delipidation and reactivation of UDPglucuronosyltransferase from rat liver

UDPglucuronosyltransferase was solubilized by treating Wistar rat liver microsomes with deoxycholate. Chromatography of this preparation on Bio-Gel P-30 resulted in extraction of 92% of phospholipids and complete loss of enzyme activity. UDPglucuronosyltransferase was reactivated by dialysing this delipidated preparation in the presence of lecithin, a mixture of liver microsomal lipids or microsomal preparations from livers of UDPglucuronosyltransferase-deficient Gunn rats. Virtually complete enzyme reactivation was obtained with regard to glucuronidation and glucosidation of bilirubin; however, the inactivation of UDPglucuronosyltransferase with p-nitrophenol as substrate was irreversible. These findings demonstrate that UDPglucuronosyltransferase with bilirubin as substrate is a lipid-requiring enzyme.     

Ehanced biliary thyroxine excretion in rats treated with pregnenolone-16alpha-carbonitrile

Normal rats were treated with pregnenolone- 16alpha - carbonitrile (PCN) 10 mg/100 g by stomach tube twice daily for 3 days. In these animals the biliary excretion of intravenously injected 125I-thyroxine (T4) was enhanced and the bile: plasma 125I ratio (B/P ratio) and the biliary clearance rate of plasma 125I-T4 was increased. Normal rats were treated with PCN for 3 days and homozygous Gunn rats for 13 days. In both groups PCN enhanced the bile flow and elevated the B/P ratios and the biliary clearance rate of plasma T4 following ip injection of 125I-T4 17 h previously. PCN-treatment had no effect on the fractions of biliary 125I present as T4-glucuronide, T4 and I- in either the normal or Gunn rats. Treatment with PCN for 10 days produced goitres in normal and Gunn rats and in normal rats elevated the serum TSH (bioassay) levels and the 17 h thyroid 131I uptake as well as the serum PB125I concentrations, without affecting stable PBI concentrations. These data indicated increased pituitary TSH release in response to increased peripheral metabolism of thyroid hormone; enhanced hormonal release from the thyroid kept pace with the accelerated peripheral loss.     

Vasopressin administration in the first month of life: effects of growth and water metabolism in hypothalamic diabetes insipidus rats

Rats homozygous for the mutant gene for diabetes insipidus (Brattleboro strain) are stunted in growth compared to rats heterozygous for the mutant gene and normal rats without the mutant gene. The hypothesis was tested that normal growth depends upon the presence of vasopressin. It was expected that replacement therapy of vasopressin rats homozygous for diabetes insipidus would make possible a normal growth rate similar to that of rats heterozygous for diabetes insipidus. Rats heterozygous and homozygous for diabetes insipidus were treated with 0.25 U (Days 0-9) and 0.5 U (Days 10-29) of vasopressin during the first month of life. During the treatment period, vasopressin significantly increased the urine osmolatities of the homozygous rats demonstrating the renal effectiveness of the vasopressin. The results showed that remedial vasopressin administration could not produce normal growth rates in homozygous rats and may be detrimental. Six weeks following vasopressin treatment, homozygous, diabetes insipidus rats which had received vasopressin had increased 24 hr water intakes and decreased urine osmolalities compared to control, homozygous rats, Heterozygous rats also had decreased urine osmolalities resulting from vasopressin six weeks after the cessation of vasopressin treatment.     

Dependence of the toxicity of the antineoplastic antibiotics rubomycin and carminomycin, on the age and sex of white mice and rats

Karminomycin was more toxic with respect to 2-6 week mice than to adult animals when administered both intravenously or orally. 5-6 week rats were more stable than adult animals to the effect of karminomycin administered intravenously or orally. Significant species sensitivity to karminomycin was noted. When administered intravenously karminomycin administered intravenously was 2 times more toxic as compared to the oral use in the experiments with adult mice and 23 times more toxic in the experiments with adult rats. LD10 and LD50 of rubomycin administerean in adult animals. LD50 of karminomycin and rubomycin administered intravenously to adult female mice was somewhat lower than that for adult male mice.     

Aiding troubled employees: the prevalence, cost, and characteristics of employee assistance programs in the United States

Bilirubin neurotoxicity can be mediated by numerous mechanisms due to its increased permeability in neuronal membranes. The present study tests the hypothesis that a prolonged bilirubin infusion modifies the N-methyl-D-aspartate (NMDA) receptor/ ion channel complex in the cerebral cortex of newborn piglets. Studies were performed in seven control and six bilirubin-exposed piglets, 2-4 d of age. Piglets in the bilirubin group received a 35 mg/kg bolus of bilirubin followed by a 4-h infusion (25 mg/kg/h) of a buffer solution containing 0.1 N NaOH, 5% human albumin, and 0.055 Na2HPO4 with 3 mg/mL bilirubin. The final mean bilirubin concentration in the bilirubin group was 495.9 +/- 85.5 mumol/L (29.0 +/- 5.0 mg/dL). The control group received a bilirubin-free buffer solution. Sulfisoxazole was administered to animals in both groups. P2 membrane fractions were prepared from the cerebral cortex. [3H]MK-801 binding assays were performed to study NMDA receptor modification. The Bmax in the control and bilirubin groups were 1.20 +/- 0.10 (mean +/- SD) and 1.32 +/- 0.14 pmol/mg protein, respectively. The value for Kd in the control brains was 6.97 +/- 0.80 nM compared with 4.80 +/- 0.28 nM in the bilirubin-exposed brains (p < 0.001). [3H]Glutamate binding studies did not show a significant difference in the Bmax and Kd for the NMDA-specific glutamate site in the two groups. The results show that in vivo exposure to bilirubin increases the affinity of the receptor (decreased Kd) for [3H]MK-801, indicating that bilirubin modifies the function of the NMDA receptor/ion channel complex in the brain of the newborn piglet. We speculate that the affinity of bilirubin for neuronal membranes leads to bilirubin-mediated neurotoxicity, resulting in either short- or long-term disruption of neuronal function.     

Experience with Sephadex gel filtration in assessing the risk of bilirubin encephalopathy in neonatal jaundice

Clinical experience with Sephadex gel filtration for evaluation of the bilirubin binding affinity of serum in neonatal jaundice is reported. In serum specimens from 362 jaundiced neonates the results of the test were in most cases in good agreement with the independently determined decision to perform exchange transfusion. In all relevant cases of the 24 with bilirubin encephalopathy the Sephadex test was strongly positive. The test was positive at lower bilirubin levels and bilirubin:albumin molar ratios in preterm as compared with term neonates, especially those in poor clinical conditions. Among term infants the test indicated an increase in bilirubin binding affinity after the fifth day of life. The Sephadex test appears to be of practical value in supplementing and refining existing criteria for exchange transfusion.     

Abnormalities in the cerebellum and brainstem in homozygous lurcher mice

The lurcher mutation induces Purkinje cell degeneration in heterozygous mice, and neonatal death in homozygous animals. Using the D6Mit16 Simple Sequence Length Polymorphic marker in F2 hybrids between AKR +/+ mice and B6+/Lc mice, homozygous lurcher fetuses and newborns as well as heterozygous and normal littermates were identified, and their brain morphology was analysed. In homozygous lurcher embryos at embryonic day 18 and neonates the cerebellum was hypotrophic, particularly in the posterior half. Purkinje cells were smaller in the whole cerebellum and showed a maturational delay. Calretinin-positive cells were less frequently observed in the depth of the vermis than in normal mice. Both Purkinje cells and the vermal calretinin-positive cells were more abnormal in fetuses at day 19 and newborn mutants than one day earlier. An abnormal number of pycnotic cells were observed in the cerebellum, especially in newborn mutants. Brainstem abnormalities were characterized by abnormal curvature, caudal displacement of the pontine gray nuclei which were located caudally along the ventral border of the superior olivary complex, a drastic decrease in Purkinje cell axons in all the vestibular nuclei and the presence of dystrophic processes in at least two calbindin-positive cell groups of the dorsal pontine region. These results show that the mutation, which is semidominant in Purkinje cells, is recessive in other cell groups of the cerebellum and brainstem. They reveal that the sequence leading to Purkinje cell death appears to be similar in homozygous and heterozygous mice, although occurring earlier and worsening more quickly in the former. Lastly, they confirm the absence of effect of the mutation on the neurons of the inferior olivary complex.     

Oral calcium phosphate: a new therapy for Crigler-Najjar disease?

BACKGROUND & AIMS: Calcium phosphate binds unconjugated bilirubin in vitro, and dietary calcium phosphate supplementation reduces the serum bilirubin level in rats with hereditary unconjugated hyperbilirubinemia (Gunn rats). The aim of this study was to evaluate the effect of oral calcium phosphate supplementation on plasma bilirubin levels in patients with Crigler-Najjar disease. METHODS: A placebo-controlled, double-blind, crossover design was used. Eleven patients, 2-42 years of age, participated. The group included 5 patients with type I disease who were all treated with phototherapy and 6 patients with type II disease who were primarily treated with phenobarbital. In addition to plasma bilirubin levels, dietary intake and urinary and fecal excretion of calcium and phosphate were evaluated. RESULTS: A modest but significant decrease in serum bilirubin was observed in patients with type I disease (18% +/- 6%, P = 0.03) but not in patients with type II disease during treatment with calcium phosphate. Urinary output of calcium and phosphate did not change during the treatment period. CONCLUSIONS: Oral calcium phosphate may be a useful adjuvant to photo-therapy in Crigler-Najjar type I disease.     

N-stearoyl-phosphatidylserine: synthesis and role in divalent-cation-induced aggregation and fusion

Ex vivo gene therapy, in which hepatocytes are harvested from mutants, retrovirally transduced with a normal gene and transplanted back into the donor, has been used for correction of inherited metabolic defects of liver. Major drawbacks of this method include limited availability of autologous hepatocytes, inefficient retroviral transduction of primary hepatocytes, and the limited number of hepatocytes that can be transplanted safely. To obviate these problems, we transduced primary hepatocytes derived from inbred bilirubin-UDP-glucuronosyl-transferase (BUGT)-deficient Gunn rats by infection with a recombinant retrovirus expressing temperature-sensitive mutant SV40 large T antigen (tsT). The immortalized cells were then transduced with a second recombinant retrovirus expressing human B-UGT, and a clone expressing high levels of the enzyme was expanded by culturing at permissive temperature (33 degrees C). At 37 degrees C, tsT antigen was degraded and the cells expressed UGT activity toward bilirubin at a level approximately twice that present in normal rat liver homogenates. For seeding the cells into the liver bed, 1 x 10(7) cells were injected into the spleens of syngeneic Gunn rats five times at 10-day intervals. Excretion of bilirubin glucuronides in bile was demonstrated by HPLC analysis and serum bilirubin levels were reduced by 27 to 52% in 40 days after the first transplantation and remained so throughout the duration of the study (120 days). None of the transplanted Gunn rats or SCID mice transplanted with the immortalized cells developed tumors.     

Monamines in brain and urine of rats with hereditary hypothalamic diabetes insipidus

Monoamine levels in brain and urine of homozygous and heterozygous diabetes insipidus (DI) rats (Brattleboro strain) were assessed. Homozygous DI rats had a higher whole brain content of serotonin than their heterozygous littermates. However, when corrected for differences in brain weight, homozygous DI also appeared to have higher brain concentrations of noradrenaline, tyrosine and GABA. The total 24 h excretion of all amines and their precursors was greater in the homozygous than in the heterozygous rats.     

Gilbert syndrome and glucose-6-phosphate dehydrogenase deficiency: a dose-dependent genetic interaction crucial to neonatal hyperbilirubinemia

Severe jaundice leading to kernicterus or death in the newborn is the most devastating consequence of glucose-6-phosphate dehydrogenase (EC 1.1.1.49; G-6-PD) deficiency. We asked whether the TA repeat promoter polymorphism in the gene for uridinediphosphoglucuronate glucuronosyltransferase 1 (EC 2.4.1.17; UDPGT1), associated with benign jaundice in adults (Gilbert syndrome), increases the incidence of neonatal hyperbilirubinemia in G-6-PD deficiency. DNA from term neonates was analyzed for UDPGT1 polymorphism (normal homozygotes, heterozygotes, variant homozygotes), and for G-6-PD Mediterranean deficiency. The variant UDPGT1 promoter allele frequency was similar in G-6-PD-deficient and normal neonates. Thirty (22.9%) G-6-PD deficient neonates developed serum total bilirubin >/= 257 micromol/liter, vs. 22 (9.2%) normals (P = 0.0005). Of those with the normal homozygous UDPGT1 genotype, the incidence of hyperbilirubinemia was similar in G-6-PD-deficients and controls (9.7% and 9.9%). In contrast, in the G-6-PD-deficient neonates, those with the heterozygous or homozygous variant UDPGT1 genotype had a higher incidence of hyperbilirubinemia than corresponding controls (heterozygotes: 31.6% vs. 6.7%, P < 0.0001; variant homozygotes: 50% vs. 14.7%, P = 0.02). Among G-6-PD-deficient infants the incidence of hyperbilirubinemia was greater in those with the heterozygous (31.6%, P = 0.006) or variant homozygous (50%, P = 0.003) UDPGT1 genotype than in normal homozygotes. In contrast, among those normal for G-6-PD, the UDPGT1 polymorphism had no significant effect (heterozygotes: 6.7%; variant homozygotes: 14.7%). Thus, neither G-6-PD deficiency nor the variant UDPGT1 promoter, alone, increased the incidence of hyperbilirubinemia, but both in combination did. This gene interaction may serve as a paradigm of the interaction of benign genetic polymorphisms in the causation of disease.     

Carcinogenic effect of chronic subcutaneous injections of 2,6-dimethylnitrosomorpholine in Sprague-Dawley rats

Sprague-Dawley rats received subcutaneous injections of 2,6-dimethylnitrosomorpholine (2,6-DMNM) at 3 different dose levels (1/5, 1/10 or 1/20 LD50) once weekly for life. Up to a 100% rate of tumors of the esophagus was detected in animals which received 1/10 and 1/20 LD50. In addition to tumors of this organ, neoplasms developed at different rates in the lungs and in the liver.     

Microdetermination of unbound bilirubin in icteric newborn sera: an enzymatic method employing peroxidase and glucose oxidase

An enzymatic assay method for the microdetermination of unbound bilirubin in newborn icteric sera is described. Unbound bilirubin is oxidized to colorless compounds by peroxidase in the presence of hydrogen peroxide derived from glucose by the mediation of glucose oxidase. In this method, the bilirubin is not significantly degraded before the addition of peroxidase, in contrast to the method using hydrogen peroxide. The oxidation rate is determined by spectrophotometry and chloroform extraction is eliminated. The unbound bilirubin concentration can be determined from the initial oxidation velocity of total bilirubin. The Michaelis constant, KM, was approximately 20 micrometer. The coefficient of variation for icteric serum determination was 4.4--6.5%. The concentration of unbound bilirubin was reduced after five days of storage at -20 degrees C. The bilirubin-albumin affinity was studied with purified albumin and adult serum. The dissociation constants were 2 x 10(-8) M and 5 x 10(-9) M, respectively, at bilirubin/albuminor molar ratios below 1.0. Clinically, serum samples from 75 icteric newborn infants were analysed, and the sera of premature infants were found to have remarkably high levels of unbound bilirubin compared to those of fullterm infants. The sera of a Rhesus immunization infant and an ABO incompatibility infant were remarkably higher than that of the nonhemolytic icteric sera. The unbound bilirubin concentration was also affected, in an in vitro study, by the addition of hemolysate.     

Acute and chronic toxicology of different staphylococcal immune preparations

The data obtained of the acute and chronic toxicity of a number of staphylococcal immune preparations are presented. The paw edema test has been shown to yield more informative results for the evaluation of acute toxicity than the method of intraperitoneal injection and LD50 determination. The paw edema test has revealed that the toxicity of corpuscular vaccine is higher than that of the preparations of water-soluble antigenic staphylococcal complexes. The chronic toxicity test has demonstrated that corpuscular vaccine, antiphagin, adsorbed toxoid and the antigenic complex have low toxicity: their multiple administration in doses exceeding the equivalent doses 30-200 times have been found to exert no influence on the increase of the weight of the animals.     

Functional integrity of hepatocyte canalicular membrane transport of taurocholate and bilirubin diglucuronide in Eisai hyperbilirubinuria rats

Eisai hyperbilirubinuria rats (EHBR) are characterized by conjugated hyperbilirubinemia, and impaired or defective excretion of bilirubin, reduced glutathione and other organic anions from hepatocytes. Hepatocyte canalicular membrane vesicles (CMV) from EHBR and normal SD rats were studied with regard to taurocholate (TC) transport driven by ATP or a membrane potential and bicarbonate-stimulated bilirubin diglucuronide (BDG) transport. ATP-dependent uptake or association of BDG with CMV was also studied in both strains of rats. No significant differences in the uptake of TC and BDG by CMV were observed. This indicates the functional integrity of the canalicular transporters for both organic anions in EHBR. Biliary excretion of taurolithocholic acid sulfate (TLCS) is defective in EHBR. However, TLCS inhibited ATP-dependent TC uptake by SD rat CMV competitively, which may be against the hypothesis that a common organic anion carrier is defective in canalicular membranes of jaundiced rats.