胆红素对新生儿脐血单核细胞凋亡的影响

目的 探讨胆红素对新生儿脐血单核细胞(CBMC)凋亡的影响.方法 采用纤维连接蛋白结合法分离新生儿CBMC.经含有不同浓度胆红素(6 mG/dl、9 mg/dl、12.9 mg/dl和18 mg/dl)的牛血清白蛋白溶液孵育1 h,再给予脂多糖(LPS,1μg/m1)刺激48 h,收集细胞.采用TUNEL法检测细胞的凋亡率.结果 LPS和低浓度(6 mg/dl)胆红素单独作用对CBMC的凋亡率均无影响.低浓度胆红素(6 mg/dl)与LPS共同作用不升高CBMC的凋亡率;较高浓度和高浓度胆红素(9 mg/dl、12.9 mg/dl、18 mg/dl)与LPS共同作用可升高CBMC的凋亡率,且这种作用随胆红索浓度的升高而增加.结论 较高浓度和高浓度胆红素可以升高CBMC的凋亡率,此种抑制作用可能与胆红素引起细胞膜破坏、Ca2+内流增多、线粒体功能障碍有关.     

Effect of calcium therapy in the sick premature infant with early neonatal hypocalcemia

Twenty-seven sick premature infants with serum calcium concentrations less than 6.0 mg/dl during the first day of age were enrolled in a prospective controlled study involving two treatment regimens--calcium given as a bolus or a drip--or no treatment. Mean total calcium concentration was 5.5 +/- 0.8 mg/dl, and ionized calcium was 3.1 +/- .3 mg/dl, with no significant difference between treatment groups. By 24 hours, in all groups total calcium had increased to greater than 6.0 mg/dl (bolus 6.5 +/- 1.1, drip 7.0 +/- 0.4, control 6.6 +/- 0.4) and ionized calcium to greater than 3.5 mg/dl (bolus 3.9 +/- 0.3, drip 3.6 +/- 0.6, control 3.6 +/- 0.3). Ionized and total calcium concentrations were significantly correlated (r = 0.562; P less than 0.001), but total calcium did not predict ionized calcium in any group. These data support the concept that, even in sick infants, early neonatal hypocalcemia is a physiologic phenomenon that may not require treatment.     

Salicylate saturation index in neonatal jaundice.

30 serum samples from premature and newborn infants with non-haemolytic hyperbilirubinaemia were analyzed to prove the accuracy of determination of albumin binding capacity for bilirubin. The salicylate method of Odell was used to determine the saturation index of albumin indicated by a decrease in optical density through displaced bilirubin. Bilirubin is stoichometrically displaced from albumin by the addition of salicylate. The values of the sautration index correspond to free binding sites. Analysis of our data demonstrated that there is no direct correlation between the saturation index (SI) and total serum bilirubin/albumin concentration quotient. Methodical errors, problems in statistics and other theoretical concepts are discussed. The salicylate method is not suitable for accurate determination of albumin binding capacity for bilirubin.     

Reduced Albumin Binding of MADDS–a Measure for Bilirubin Binding–in Sick Children

ABSTRACT. The reserve albumin concentration for binding of MADDS (monoacetyldiaminodiphenyl sulphone) in plasma is used as a measure of the reserve albumin concentration for binding of unconjugated bilirubin. The aim of the present study was to investigate whether a reduction in the reserve albumin concentration for binding of MADDS could exist in sick children after 5 months of age, where the bilirubin binding properties of the albumin has reached the adult level. The material included 75 children, 1-15 years of age with mild infections, servere bacterial infections, acute viral hepatitis, chronic hepatic diseases or uraemia, and a control group of 22 healthy children. The reserve albumin concentration was significantly lower in children with severe bacterial infections, acute viral hepatitis, and uraemia, than in healthy children ( p <0.01), while the reserve albumin concentration in children with mild infections and chronic hepatic diseases did not differ significantly from that of the control group ( p > 0.05). The total albumin concentration in plasma in either of the groups of sick children did not differ significantly from that of the healthy children. The plasma concentration of unconjugated bilirubin was elevated in the group of children with acute viral hepatitis, but not enough to influence the concentration of reserve albumin for binding of MADDS to a significant degree. The reserve albumin concentration was significantly lower in children with acute viral hepatitis than in children with severe bacterial infections ( p <0.05).     

Reduced albumin binding of MADDS--a measure for bilirubin binding--in sick children

The reserve albumin concentration for binding of MADDS (monoacetyldiaminodiphenyl sulphone) in plasma is used as a measure of the reserve albumin concentration for binding of unconjugated bilirubin. The aim of the present study was to investigate whether a reduction in the reserve albumin concentration for binding of MADDS could exist in sick children after 5 months of age, where the bilirubin binding properties of the albumin has reached the adult level. The material included 75 children, 1-15 years of age with mild infections, severe bacterial infections, acute viral hepatitis, chronic hepatic diseases or uraemia, and a control group of 22 healthy children. The reserve albumin concentration was significantly lower in children with severe bacterial infections, acute viral hepatitis, and uraemia, than in healthy children (p less than 0.01), while the reserve albumin concentration in children with mild infections and chronic hepatic diseases did not differ significantly from that of the control group (p greater than 0.05). The total albumin concentration in plasma in either of the groups of sick children did not differ significantly from that of the healthy children. The plasma concentration of unconjugated bilirubin was elevated in the group of children with acute viral hepatitis, but not enough to influence the concentration of reserve albumin for binding of MADDS to a significant degree. The reserve albumin concentration was significantly lower in children with acute viral hepatitis than in children with severe bacterial infections (p less than 0.05).     

EFFECTS OF STABILIZERS IN PREPARATIONS OF HUMAN SERUM ALBUMIN ON THE SERUM BILIRUBIN IN GUNN RATS

ABSTRACT. Commercially available preparations of human serum albumin (HSA) containing stabilizers (i.e. 16 mmol/I Na caprylate plus 16 mmol/I Na N-acetyl- dl -tryptophan) were injected either s.c., i.p. or i.v. into homozygous infant Gunn rats. 30 min and 3 hours after s.c. injection, a serum bilirubin decline which surpassed dilution by the injected volume could be ascertained. It was mainly caused by N-acetyl- dl -tryptophan since s.c. injections of appropriate amounts of this substance alone or a mixture of both components of the stabilizer without HSA brought about similar results. HSA without these stabilizers had not such an effect. It is postulated that under these conditions Na N-acetyl- dl -tryptophanate displaced bilirubin from albumin bonds. It became obvious that after s.c. injection equilibration of HSA between skin and plasma was delayed, whereas Na N-acetyl- dl -tryptophan was rapidly transported to the blood. As for Na caprylate, a displacing effect of short duration could not be excluded by the experimental arrangement used, since the metabolism of the substance in the rat is very fast. When HSA and the stabilizers entered the plasma simultaneously (i.v. injection) no effect on serum bilirubin concentration could be proved 30 min and 3 hours later. All the bilirubin and the Na N-acetyl- dl -tryptophan present in the plasma at that time can be bound to the large amount of albumin which is directly given into the circulation of the animal. 30 min after i.p. injection of HSA preparations containing stabilizers a small decrease of serum bilirubin concentration could be recognized. It was less pronounced and less persisting than after s.c. injection. Probably equilibration of HSA between peritoneum and plasma went on faster than between skin and plasma. Only for a short period a lack of albumin binding sites in the plasma of the rat pointed to a surplus of Na N-acetyl- dl -tryptophan.     

Management of hyperbilirubinemia and prevention of kernicterus in 20 patients with Crigler-Najjar disease

We summarize the treatment of 20 patients with Crigler-Najjar disease (CND) managed at one center from 1989 to 2005 (200 patient-years). Diagnosis was confirmed by sequencing the UGTA1A gene. Nineteen patients had a severe (type 1) phenotype. Major treatment goals were to maintain the bilirubin to albumin concentration ratio at <0.5 in neonates and <0.7 in older children and adults, to avoid drugs known to displace bilirubin from albumin, and to manage temporary exacerbations of hyperbilirubinemia caused by illness or gallstones. A variety of phototherapy systems provided high irradiance over a large body surface. Mean total bilirubin for the group was 16+/-5 mg/dl and increased with age by approximately 0.8 mg/dl per year. The molar ratio of bilirubin to albumin ranged from 0.17 to 0.75 (mean: 0.44). The overall non-surgical hospitalization rate was 0.12 hospitalizations per patient per year; one-half of these were for neonatal hyperbilirubinemia and the remainder were for infectious illnesses. Ten patients (50%) underwent elective laproscopic cholecystectomy for cholelithiasis. No patient required invasive bilirubin removal or developed bilirubin-induced neurological damage under our care. Visual acuity and color discrimination did not differ between CND patients and age-matched sibling controls. Four patients treated with orthotopic liver transplantation were effectively cured of CND, although one suffered significant transplant-related complications.Conclusions. While patients await liver transplantation for CND, hyperbilirubinemia can be managed safely and effectively to prevent kernicterus. Lessons learned from CND can be applied to screening and therapy of non-hemolytic jaundice in otherwise healthy newborns.     

RESERVE ALBUMIN AND BILIRUBIN TOXICITY INDEX IN INFANT SERUM

ABSTRACT. Reserve albumin concentration (the concentration of albumin available for binding of unconjugated bilirubin) was determined in 95 sera from 76 subjects by dialysis with ¹⁴C-monoacetyl diamino diphenyl sulfone (MADDS). An index, I of bilirubin toxicity in the plasma was calculated for each subject, based on the bilirubin and reserve albumin concentrations, the affinity of bilirubin for serum albumin, and the pH-dependent solubility of bilirubin in the plasma. The values of reserve albumin and of I varied significantly with gestational age, clinical condition (whether sick or well), and serum bilirubin level. The value of reserve albumin was decreased and I was increased in association with clinical factors (e. g., hyperbilirubinemia, hypoxia, acidosis, or sepsis) recognized as increasing the risk for bilirubin encephalopathy. The lowest values of reserve albumin and the highest values of I were found in the least mature and sickest infants.     

Use of intravenous lipid and hyperbilirubinemia in the first week.

Serum triglycerides, free fatty acids, unconjugated bilirubin, and albumin were evaluated in 40 neonates receiving 0.5-3.5 g/kg/day of a 50/50 soybean-safflower lipid emulsion infused during 18 h. The purpose of the study was to evaluate lipid tolerance and unconjugated hyperbilirubinemia according to our total parenteral nutrition protocol, which initiates lipid on postnatal day 4. Mean serum triglycerides and free fatty acids were within the range of prelipid infusion values at all dosages, and no statistically significant differences were noted between very-low-birth-weight neonates and those greater than 1,500 g birth weight. Mean free fatty acid:albumin molar ratio was less than 1.0 at all dosages and no individual patient values exceeded a ratio of 3.0. Mean peak serum unconjugated bilirubin of 5.8 mg/dl on postnatal day 3 was stable or fell the next 10 days of lipid-inclusive total parenteral nutrition. Initiating intravenous lipid on the 4th postnatal day at 0.5 g/kg/day and increasing at 0.5 g/kg/day increments at the end of the 1st postnatal week appears to be tolerated well. However, 5% of serum triglyceride levels exceeded 200 mg/dl. Therefore, in view of the unpredictability of a given patient's tolerance to lipid infusion, there should be monitoring for lipemia.     

Determination of bilirubin in capillary plasma by a direct photometric method (DPM, bilirubinometer) and the chemical determination of bilirubin in the bilirubin determination (2,5-dichlorophenyldiazonium method) in serum of venous blood samples

The determination of bilirubin in serum was performed by the 2.5-dichlorphenyldiazonium method (DPD) and in capillary plasma by the direct photometric method (DPM). Both methods showed a good precision and accuracy. The investigation was carried out in 135 samples with a bilirubin concentration up to 25 mg/dl. The comparison of the two methods in 62 samples with a bilirubin concentration up to 10 mg/dl showed a correlation coefficient of r = 0.862 and in 73 samples with a bilirubin concentration between 10 and 25 mg/dl a correlation coefficient of r = 0.893. In 29 cases (21.5%) we found differences between the two methods of 1.5-4.0 mg/dl. Most of them were in the critical higher range. Discussion of the DPD and DPM methods.     

Relationship of unbound bilirubin concentration to reserve albumin-binding concentration for bilirubin in human neonatal plasma

We examined the relationship of plasma unbound bilirubin concentration and reserve albumin-binding concentration for bilirubin in a sample of 545 neonates. Plasma unbound bilirubin concentration and total bilirubin-binding concentration were determined with the peroxidase assay. Contrary to published reports, we found that plasma unbound bilirubin concentration and plasma reserve albumin for bilirubin-binding concentration are highly correlated (r = -0.706; p less than 0.001) and that the relationship between these two parameters is dependent upon the total bilirubin-binding concentration. That these measured parameters correlate in the same manner as predicted for free bilirubin and free albumin by the law of mass action, suggests that these measurements may be meaningful.     

The importance of free bilirubin acid salt in bilirubin uptake by erythrocytes and mitochondria

The binding of bilirubin to tissue was studied using adult human erythrocytes and rat liver mitochondria. Tissues were incubated with varying bilirubin-albumin molar ratios, varying albumin concentrations of a given bilirubin-albumin molar ratio, and varying pH. Bilirubin binding by tissue was reversible and stoichiometric with the concentration of the free (nonalbumin bound) bilirubin acid salt (bilirubin monovalent anion). Minimal binding of the bilirubin dianion, the predominant state of bilirubin in plasma, was also suggested. The observations support the "free bilirubin theory" where tissue and albumin compete for binding the body's bilirubin pool. Binding to tissue, however, is not determined by the free bilirubin concentration, but by the concentration of the pH dependent subfraction, the free bilirubin acid salt. Tissue binding and toxicity of bilirubin may result from the surfactant properties of the monovalent anion.     

Bilirubin displacing effect of stabilizers added to injectable preparations of human serum albumin

Stabilizers added to preparations of human serum albumin before heat treatment were tested for bilirubin displacing effect, using the peroxidase method. It was found that N-acetyltryptophan and sodium caprylate displace bilirubin from its complex with human serum albumin in vitro. The quantitative findings were used for a rough estimate of the effect of these substances on the free bilirubin concentration in blood plasma, expected when stabilized albumin preparations are given intravenously for prevention of kernicterus. The calculated effect is a delay of the decrease of free bilirubin concentration, or even a temporary increase. Sodium mandelate displaces less strongly.     

Increase in bilirubin binding to albumin with correction of neonatal acidosis.

Twenty-six serial measurements of free bilirubin concentration and apparent association constant of bilirubin for albumin (Ka) at a bilirubin: albumin molar ratio of 0.8 were performed and compared with baseline values in 11 newborn infants with acidosis before treatment and during recovery from acidosis. When arterial pH was corrected from 7.12 +/- 0.02 (Mean +/- S.EM.) to 7.34 +/- 0.02, there was a significant decrease in serum free bilirubin concentration and a significant increase in the Ka at molar ratio 0.8. The data offer in vivo evidence that correction of acidosis in the neonate results in an improvement of the apparent bilirubin binding affinity of albumin.     

BILIRUBIN DISPLACING EFFECT OF STABILIZERS ADDED TO INJECTABLE PREPARATIONS OF HUMAN SERUM ALBUMIN

Abstract Stabilizers added to preparations of human serum albumin before heat treatment were tested for bilirubin displacing effect, using the peroxidase method. It was found that N-acetyltryptophan and sodium caprylate displace bilirubin from its complex with human serum albumin in vitro. The quantitative findings were used for a rough estimate of the effect of these substances on the free bilirubin concentration in blood plasma, expected when stabilized albumin preparations are given intravenously for prevention of kernicterus. The calculated effect is a delay of the decrease of free bilirubin concentration, or even a temporary increase. Sodium mandelate displaces less strongly.     

Randomized trial of prophylactic phototherapy in the infant with very low birth weight

Twenty-two preterm infants (birth weight 850 +/- 220 gm) were randomly assigned to receive phototherapy either soon after birth or after the serum bilirubin concentration reached 5 mg/dl. Infants receiving prophylactic phototherapy were placed under lights at a significantly earlier age and lower serum bilirubin concentration than infants in the routine group (P less than 0.001). There was no significant difference between groups in peak serum bilirubin concentration, age at which it peaked, rate of rise in serum bilirubin concentration, or serum bilirubin concentration at any time during the study. Infants assigned to the prophylactic phototherapy group were under lights for a significantly longer time than those in the routine group (P less than 0.05). There was a significant rise in both configurational and structural photo-isomers (P less than 0.005) independent of serum bilirubin concentration after phototherapy in all patients. These data suggest that the clinical course of hyperbilirubinemia is not altered in infants with very low birth weight receiving prophylactic phototherapy compared with infants with phototherapy begun at a bilirubin concentration of 5 mg/dl.     

INCREASE IN BILIRUBIN BINDING TO ALBUMIN WITH CORRECTION OF NEONATAL ACIDOSIS

Abstract. Twenty-six serial measurements of free bilirubin concentration and apparent association constant of bilirubin for albumin (Ka) at a bilirubin: albumin molar ratio of 0.8 were performed and compared with baseline values in 11 newborn infants with acidosis before treatment and during recovery from acidosis. When arterial pH was corrected from 7.12±0.02 (Mean±S.E.M.) to 7.34±0.02, there was a significant decrease in serum free bilirubin concentration and a significant increase in the Ka at molar ratio 0.8. The data offer in vivo evidence that correction of acidosis in the neonate results in an improvement of the apparent bilirubin binding affinity of albumin.     

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.     

Evaluation of the bilirubinometer Hico-Bilimat A 700]

The Hico-Bilimeter A 700 is a simple filter photometer for the determination of total bilirubin in neonatal serum, reading the absorbance of the undiluted sample at two wavelengths. Precision of the measurements is good, with linearity acceptable up to a concentration of 18 mg/dl. Compared to the values obtained with the reference method the results below 18 mg/dl are 16% too high on average; above 18 mg/dl the scatter of results is markedly increased. Even a moderate hemolysis shows a distinct influence on the measurements. Using the instrument tested no reliable results could be obtained, especially in hemolytic samples.     

Experimental bilirubin encephalopathy: importance of total bilirubin, protein binding, and blood-brain barrier

The cause of bilirubin encephalopathy has been variously ascribed to elevated total serum bilirubin concentration, high free bilirubin levels (or impaired albumin binding), and disruption of the blood-brain barrier. An experimental rat model for acute bilirubin encephalopathy was developed in which these three factors could be varied independently. Osmotic opening of the blood-brain barrier in the right hemisphere was produced by infusing a hypertonic arabinose solution into the right carotid artery. The total bilirubin level and bilirubin binding state were varied by adjusting the amount of bilirubin infused intravenously and/or by infusing human serum albumin. Brain electrical activity (EEG) served as an indicator of developing encephalopathy. Neither staining nor EEG changes occurred if the blood-brain barrier remained intact. Bilirubin staining without EEG evidence of encephalopathy sometimes occurred when the blood-brain barrier was open. Discriminant analysis showed that EEG changes were best predicted by the degree of blood-brain barrier opening (as indicated by brain bilirubin content) and by the quality of serum bilirubin binding. Serum total bilirubin concentration was not an important discriminator of encephalopathy.