A novel inhibitor of beta-glucuronidase: L-aspartic acid.

Infants who consume casein hydrolysate formula have been shown to have lower neonatal jaundice levels than infants who consume routine formula or breast milk. Because casein hydrolysate has been shown to contain a beta-glucuronidase inhibitor, one possible mechanism to explain this finding is blockage of the enterohepatic circulation of bilirubin by a component of the formula. The aim of this research was to identify the source of the beta-glucuronidase inhibition in hydrolyzed casein. A beta-glucuronidase inhibition assay and measurements of physical and kinetic parameters were used to analyze the components of hydrolyzed casein and infant formulas. Kinetic studies used purified beta-glucuronidase. The L-aspartic acid in hydrolyzed casein accounts for the majority of the beta-glucuronidase inhibition present. Kinetic studies indicate a competitive inhibition mechanism. L-aspartic acid is a newly identified competitive inhibitor of beta-glucuronidase.

Transcutaneous bilirubin measurement: a multicenter evaluation of a new device.

OBJECTIVES: The early discharge of neonates from hospitals makes transcutaneous measurement of total bilirubin concentration a useful tool to monitor neonatal jaundice. The objectives of this study were to determine whether 1) transcutaneous bilirubin (TcB) measurement, as performed using BiliCheck (BC), correlates with total serum bilirubin (TSB) levels, measured with standard laboratory methods and with high-pressure liquid chromatography (HPLC-B); 2) infant race, gestational age, postnatal age, or body weight interferes with the measurement of TcB levels in newborn infants; 3) the variability of the TcB measurement is comparable to the variability of TSB measurements; and 4) TcB measurements obtained from the forehead (BCF) and sternum (BCS) generate comparable results. STUDY DESIGN: Newborn infants who were <28 days and >30 weeks' gestational age and who underwent tests for TSB as part of their normal care in 6 different European hospitals were studied. A total of 210 infants were enrolled in the study, 35 at each site. Near simultaneous (within +/- 30 minutes) blood collection for TSB and BCF and BCS measurements were performed. TSB levels were determined by the serum bilirubin method in use at each site, and all HPLC-B determinations were made at the same, independent laboratory. RESULTS: The study group consisted of 140 white, 31 Asian, 14 Hispanic, 9 African, and another 16 newborns of different races. The correlation coefficient (r) between BCF and HPLC-B was 0.890 (95% confidence interval = 0.858-0.915). BCF and BCS generated similar results (r value = 0.890 for BCF and 0.881 for BCS), even if BCS slightly overestimated (mean error = -0.04 mg/dL) and BCF slightly underestimated (mean error = 0.96 mg/dL) in comparison with HPLC-B. Analysis of covariance demonstrated that BC accuracy was independent of race, birth weight, gestational age, and postnatal age of the newborn. Receiver operating characteristic curves were evaluated for BCF and TSB, each compared with HPLC-B. With the use of a cutoff point for HPLC-B of 13 mg/dL (222 micromol/L) and a cutoff of 11 mg/dL on the BCF and TSB, similar sensitivity/specificity (93%/73% for BCF, 95%/76% for TSB) were observed. The use of a cutoff point for HPLC-B of 17 mg/dL (290 micromol/L) and 14 mg/dL (240 micromol/L) for BCF and TSB also produced similar sensitivity/specificity (90%/87% for the BC and 87%/83% for TSB). CONCLUSIONS: Because the correlation coefficient for HPLC-B and BCF is very similar to that found for HPLC-B and laboratory TSB, BC could be used not only as a screening device but also as a reliable substitute of TSB determination. At higher levels of TSB, in which phototherapy and/or exchange transfusion might be considered, BC performed slightly better than the laboratory. The accuracy and precision of the TcB measurement in this study was observed to be comparable to the standard of care laboratory test.

Use of maternal plasma level of zinc-coproporphyrin in the prediction of intrauterine passage of meconium: a pilot study.

OBJECTIVE: To evaluate the usefulness of maternal plasma zinc-coproporphyrin (ZCP) level as a marker for intrauterine passage of meconium. METHODS: A pilot study consisting of 10 pregnancies with meconium-stained amniotic fluid and 10 pregnancies with clear amniotic fluid was used. The corresponding plasma and amniotic fluid levels of ZCP were measured using spectrofluorometry. ZCP levels in plasma and amniotic fluid were compared between the two groups and the relation between plasma and amniotic fluid ZCP levels in the clear and meconium-stained groups was assessed using Spearman rank-order correlation. RESULTS: Mean amniotic fluid ZCP was significantly higher in the meconium-stained amniotic fluid as compared to the clear amniotic fluid group. Although mean plasma ZCP levels were higher in the meconium-stained amniotic fluid group, this difference was not statistically significant. There was no significant correlation between plasma ZCP levels and amniotic fluid ZCP, but we could categorize patients according to plasma ZCP levels into four categories with different risks for having meconium-stained amniotic fluid. CONCLUSIONS: Plasma ZCP might be a promising test for prediction of intrauterine passage of meconium in high-risk patients if confirmed by larger studies. The implications of this prediction on management remain unknown.

Noninvasive measurement of total serum bilirubin in a multiracial predischarge newborn population to assess the risk of severe hyperbilirubinemia.

BACKGROUND: Jaundice in near-term and term newborns is a frequent diagnosis that may prompt hospital readmission in the first postnatal week. Hyperbilirubinemia, when excessive, can lead to potentially irreversible bilirubin-induced neurotoxicity. Predischarge risk assessment (at 24-72 hours of age) for subsequent excessive hyperbilirubinemia is feasible by a laboratory-based assay of total serum bilirubin (TSB). Hypothesis. Noninvasive, transcutaneous, point-of-care measurement of transcutaneous bilirubin (TcB) predischarge by multiwavelength spectral analysis, using a portable BiliCheck device (SpectRx Inc, Norcross, GA), is clinically equivalent to measurement of TSB in a diverse, multiracial term and near-term newborn population and predictive of subsequent hyperbilirubinemia. METHODOLOGY: We evaluated a hand-held device that uses multiwavelength spectral reflectance analysis to measure TcB (BiliCheck). The study population (490 term and near-term newborns) was racially diverse (59.1% white, 29.5% black, 3.46% Hispanic, 4.48% Asian, and 3.46% other) and was evaluated at 2 separate institutions using multiple (11) devices. The postnatal age ranged from 12 to 98 hours and the ranges of birth weights and gestational ages were 2000 to 5665 g and 35 to 42 weeks, respectively. All transcutaneous evaluations were performed contemporaneously and paired with a heelstick TSB measurement. All TSB assays were performed by high performance liquid chromatography, as well as by diazo dichlorophenyldiazonium tetrafluoroborate techniques. RESULTS: TSB values ranged from .2 to 18.2 mg/dL (mean +/- standard deviation: 7.65 +/- 3.35 mg/dL). The overall correlation of TSB (by high performance liquid chromatography technique) to TcB (by BiliCheck devices) was linear and statistically significant (r =.91; r(2) =.83; TcB =.84; TSB = +.75; standard error of regression line = 1.38; P <.001; n = 490 infants; 1788 samples). Similar regression statistics were evident in subset populations categorized by race (white: r =.91 [n = 289 infants]; black: r =.91 [n = 145 infants]) as well as by gestation (term: r =. 91 [n = 1625 samples]; near-term: r =.89 [n = 163 samples]). Intradevice precision was determined to be.59 mg/dL (2-3 measurements per infant with 1 device; n = 210 infants; 510 samples in a separate subset). Interdevice evaluation of 11 devices determined the precision to be.68 mg/dL (2-4 devices used for measurements per patient). In 23 of 419 of the study population infants who were in the 24- to 72-hour age range, the predischarge TSB values designated them to be at high risk for subsequent excessive hyperbilirubinemia (above the 95th percentile track on the hour-specific bilirubin nomogram). For these infants, the paired BiliCheck TcB values were all above the 75th percentile track (negative predictive value = 100%; positive predictive value = 32. 86%; sensitivity = 100%; specificity = 88.1%; likelihood ratio = 8. 43). CONCLUSIONS: Our data demonstrate the accuracy and reproducibility of the predischarge BiliCheck measurements in term and near-term newborn infants of diverse races and ethnicities. Infants with predischarge BiliCheck values above the 75th percentile of hour-specific TSB values on the bilirubin nomogram may be considered to be at high risk for subsequent excessive hyperbilirubinemia. Further studies are needed to assess the efficacy of this technique in preterm infants, those undergoing phototherapy, and those with TSB values of >/=15 mg/dL (>/=256 micromol/L).

Neonatal jaundice and diet.

OBJECTIVE: To determine whether an earlier observation, that infants fed a casein-hydrolysate formula (Nutramigen) have lower neonatal jaundice levels than those fed standard formulas, would be repeated in a larger independent group of infants with more frequent measurements and more rigorous statistical analysis. DESIGN: Newborn infants were fed human milk, a standard whey-predominant formula (Enfamil), or Nutramigen (n = 20 for each group) during the first 3 weeks of life. Transcutaneous jaundice index was measured daily for the first week of life and every 2 to 3 days thereafter, using a noninvasive jaundice meter. Linear regression models of the data were constructed, validated, and compared statistically. SETTING: General community hospital with subsequent home visitation. PARTICIPANTS: Healthy, term newborn infants selected by convenience, based on time of birth. INTERVENTION: Infants were exclusively fed human milk, Enfamil, or Nutramigen. Formulas were randomly assigned. MAIN OUTCOME MEASURE: Jaundice index, a transcutaneous measurement of jaundice. RESULTS: The jaundice index differed significantly among the 3 groups. Paired comparisons showed that the jaundice index of the Nutramigen group was significantly lower than that of the Enfamil group (on days 6-16) and the human milk group (on days 3-20). The jaundice index of the Enfamil-fed group was significantly lower than that of the human milk group on days 13 to 19. CONCLUSIONS: Jaundice levels are lower in neonates fed Nutramigen rather than Enfamil and both these groups have lower jaundice levels than breast-fed infants.

Gilbert syndrome accelerates development of neonatal jaundice.

OBJECTIVE: Gilbert Syndrome (GS), associated with unconjugated hyperbilirubinemia and decreased bilirubin UDP-glucuronosyltransferase activity, is usually diagnosed after puberty. The role of GS in neonatal jaundice is unknown. This study tested the hypothesis that a recently identified molecular marker for GS (a TA insertion in the promoter of UGT1A, the gene encoding bilirubin UDP-glucuronosyltransferase) is associated with neonatal jaundice. STUDY DESIGN: Transcutaneous jaundice index was measured shortly after birth and daily for the first week of life in 151 healthy infants. Genomic DNA was isolated from blood or buccal brushings, and the UGT1A promoter was amplified by the polymerase chain reaction to yield 90 (A[TA]6TAA, normal) or 92 (A[TA]7TAA, GS) base pair products. Statistical analysis used Kruskal-Wallis, Wilcoxon, and Fisher's exact tests. RESULTS: Nineteen (13%) subjects were homozygous for the A(TA)7TAA polymorphism associated with GS. The A(TA)7TAA homozygotes had a greater increase in jaundice index during the first 2 days of life than heterozygotes or A(TA)6TAA homozygotes. CONCLUSION: Although peak jaundice levels did not differ among groups, newborn infants with the molecular marker for GS have an accelerated increase in neonatal jaundice during the first 2 days of life.

Inhibition of beta-glucuronidase by casein hydrolysate formula.

BACKGROUND: A casein hydrolysate infant formula has been shown to be associated with lower levels of neonatal jaundice than are standard infant formulas. Because beta-glucuronidase is related to neonatal jaundice, this study examined the effect of a casein hydrolysate formula on beta-glucuronidase. METHODS: Beta-glucuronidase activity was measured with or without added dietary components. The beta-glucuronidase sources used were meconium, breast milk, and the purified bovine liver enzyme. The dietary components assayed for their effect on beta-glucuronidase activity included casein hydrolysate formula (Nutramigen), whey-predominant formula (Enfamil), breast milk; enzymatically hydrolyzed casein, and other constituents of the casein hydrolysate formula. Stool samples of 6-day-old infants, who were exclusively fed one of the two formulas or breast milk, were also assayed for inhibition of beta-glucuronidase. RESULTS: Only Nutramigen, enzymatically hydrolyzed casein, and stool from Nutramigen-fed infants consistently demonstrated significant inhibition of beta-glucuronidase activity, ranging from 45% to 85% of that in controls. The inhibition of beta-glucuronidase in purified bovine liver demonstrates a dose response in a pH range from 4 to 7.3. CONCLUSIONS: Hydrolyzed casein contains a beta-glucuronidase inhibitor that, in casein hydrolysate-fed infants, persists after passage through the digestive tract. These data are consistent with the possibility that inhibition of beta-glucuronidase is a mechanism by which infants fed casein hydrolysate have lower jaundice levels than infants fed routine formulas or breast milk. Further study of this mechanism is needed.

Bilirubin metabolism and kernicterus.

Neonatal jaundice continues to be a common problem. Kernicterus, although rare, continues to be a very real concern in both full-term and preterm infants. The diagnosis of kernicterus requires not only bilirubin staining in a characteristic pattern in the brain but also neuronal damage. With careful pathologic evaluation, kernicterus should be distinguishable from the brain damage associated with asphyxia and hypoxia. Early hospital discharge is a risk factor for the development of kernicterus. Combining the use of traditional phototherapy from above and a fiberoptic blanket from below has improved the effectiveness of phototherapy. Clinical trials with SnMP as an inhibitor of heme oxygenase appear encouraging; no adverse effects were noted, except for mild, occasional photosensitization manifest by erythema in babies receiving phototherapy. One theoretical toxicity of inhibitors of heme oxygenase involves the recent observation that carbon monoxide (CO) is a neurotransmitter in certain regions of the brain, possibly comparable to nitric oxide (NO), and the consequences of such inhibition are unknown. More research is needed to improve our understanding about the entry of bilirubin into the brain, the predilection of bilirubin for certain brain regions, and the cytotoxicity of bilirubin. In the United States, there is currently no generally accepted method to predict hyperbilirubinemia or kernicterus. Brain stem auditory evoked responses and MRI can both be used effectively to monitor the effects of severe hyperbilirubinemia.

The effect of diet on feces and jaundice during the first 3 weeks of life.

This study examined the relationship between fecal output and neonatal jaundice in infants exclusively fed either human milk or one of three infant formulas (whey predominant, Enfamil; casein predominant, 3305H; and casein hydrolysate, Nutramigen; all from Mead Johnson, Evansville, IN). Stool output was quantitated during the first 3 weeks of life. Jaundice was assessed by measuring serum bilirubin level and transcutaneous jaundice index. In general, after the fourth day, breast-fed infants produced lower-weight individual wet and dry stools than formula-fed infants. Cumulative wet and dry stool output was also lowest in the breast-fed infants during this time. After the first week, breast-fed infants had a higher stooling frequency than formula-fed infants. The jaundice indexes of the four groups differed significantly on all days after day 3, with highest levels in breast-fed infants and lowest levels, for unknown reasons, in those fed casein hydrolysate. The jaundice index of those fed casein hydrolysate was significantly lower than that of the other formula-fed infants on days 10-18. In the breast-fed group the decrease from day 3 to day 21 in both serum bilirubin level and the jaundice index was positively correlated with both the 21-day total wet and total dry cumulative stool weights. It is concluded that the quantity of stool excreted is related to decreases in serum bilirubin levels in infants fed human milk.

Erosions of the angelchik prosthesis in pediatric-sized developmentally disabled patients.

We reviewed case histories of 40 pediatric-sized developmentally disabled patients who had previously participated in a study comparing the Nissen fundoplication with the Angelchik prosthesis for the surgical treatment of severe gastroesophageal reflux. Five of these patients had experienced erosions of the prosthesis into the gastrointestinal tract. These erosions were diagnosed between 2 years and 2 years 8 months following surgical insertion of the device. Erosions were associated with a variety of symptoms including vomiting, increasing discomfort, melena, anemia, coffee ground gastric residuals, and repeated small bowel obstructions. In no case was erosion associated with the development of peritonitis. Despite the documented advantages of the Angelchik prosthesis, the 12.5% erosion rate in this patient population is excessive. We recommend that use of the Angelchik prosthesis is not advisable in pediatric-sized developmentally disabled patients.

Verotoxic Escherichia coli in human disease.

Verotoxin-producing E. coli (most frequently E. coli O157) have been implicated in the pathogenesis of diarrhea, hemorrhagic colitis, hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. Cattle, meat products, and other sources have been found to harbor these organisms. Isolation of E. coli O157:H7 on MacConkey-sorbitol agar is diagnostic, yet the bacteria are difficult to detect after the first week of infection. Enzyme linked immunosorbent assays (ELISAs) can detect verotoxin in fecal filtrates in the absence of viable bacteria. Serologic evidence has also been used to support the diagnosis of verotoxin-associated infection. Evidence supporting the etiologic role of different verotoxins is reviewed. Treatment remains supportive since the use of antibiotics and antimotility agents can lead to poorer outcomes. Recommendations for prevention are presented.

Excremental studies in human neonates. Identification of zinc coproporphyrin as a marker for meconium.

Zinc coproporphyrin, isomers I and III (I:III ratio, approximately 8:1), was identified in human meconium using high-performance liquid chromatography, ultraviolet-visible absorbance spectroscopy, and emission fluorescence spectroscopy. The high levels of zinc coproporphyrin in the first stools of life decrease steadily as meconium is evacuated and reach a plateau at comparatively negligible levels after complete evacuation of meconium. Zinc coproporphyrin can be used as a marker for human meconium.

High-performance liquid chromatographic analysis of bile pigments as their native tetrapyrroles and as their dipyrrolic azosulfanilate derivatives.

A reversed-phase high-performance liquid chromatographic (HPLC) analysis of bile pigments is described that provides baseline separation of the major bilirubin conjugates found in bile. The advantage of the technique is that the bile pigments can be analyzed directly as their native tetrapyrroles without prior solvent extractions or derivatization. The use of ammonium acetate in place of sodium salts permits preparative isolation and lyophilization of the pigments for mass spectroscopy. The derivatization of the pigments as their dipyrrolic azosulfanilates with subsequent HPLC analysis demonstrates baseline separation of the endo- and exovinyl azodipyrroles and allows identification of that half of the tetrapyrrole which contains the conjugate in the instances of monoglycosides.

The effect of saccharolactone on rat intestinal absorption of bilirubin in the presence of human breast milk.

Beta-glucuronidase hydrolyzes glucuronic acid from bilirubin glucuronides. The unconjugated bilirubin that results is more readily absorbed from the intestine. Human breast milk has significant beta-glucuronidase activity, and it has been suggested that the milk may play an etiologic role in the hyperbilirubinemia commonly seen in breast-fed infants. To test whether breast-milk can facilitate intestinal bilirubin absorption, pairs of rats were fitted with bile duct and duodenal catheters. One rat of each pair received an intraduodenal infusion of rat bile plus breast-milk; the other rat received a similar amount of bile and milk plus the beta-glucuronidase inhibitor saccharolactone. Rats receiving saccharolactone excreted significantly less bilirubin in their bile, suggesting that inhibition of beta-glucuronidase decreased intestinal absorption of bilirubin. These findings were not seen in similar experiments when saline was substituted for human breast-milk.

Bile and stone analysis in two infants with brown pigment gallstones and infected bile.

Two infants under 3 mo of age who presented with obstructive jaundice secondary to cholelithiasis are reported. Neither infant had any congenital anatomic abnormality of the biliary tract leading to stasis, yet both had cultures of gallbladder bile that grew abundant bacteria. In both, recovery of gallbladder bile and sludge or actual stones allowed a detailed analysis of bile and stone composition. Bile was not saturated with cholesterol. In both cases, unconjugated bilirubin accounted for a large percentage of the total bile biliary pigments measured, and stercobilin was present in gallbladder bile. Bile beta-glucuronidase activity was higher when measured at the optimal pH of bacterial rather than tissue beta-glucuronidase. Analysis of stone morphology and composition showed characteristics of brown pigment gallstones with a layered appearance and the presence of calcium palmitate. This is the first report of detailed bile and stone analysis in infants and supports the hypothesis that brown pigment gallstones form spontaneously in infants who have bacterial infections in the biliary tract.

Alpha 1-antitrypsin deficiency and the PiMS phenotype: case report and literature review.

We describe a premature infant with cholestatic liver disease and protease inhibitor MS phenotype. This infant demonstrated an abnormally low serum alpha 1-antitrypsin concentration. Liver histologic studies revealed diastase-resistant, periodic acid-Schiff-positive globules inside hepatocytes. Immunoperoxidase staining for alpha 1-antitrypsin was positive. Electron microscopy showed amorphous material in the dilated lumina of the endoplasmic reticulum. These findings are characteristic of alpha 1-antitrypsin deficiency. We suggest that this usually nonpathologic phenotype resulted in cholestatic liver disease because of the cumulative effect of several cholestatic conditions.

Ascites complicating ventriculoperitoneal shunts.

Ventriculoperitoneal shunts are currently a standard therapy for obstructive hydrocephalus. These shunts are associated with a variety of abdominal complications, one of which is the development of ascites. We report an 11-year-old girl with a ventriculoperitoneal shunt in whom a low-grade peritoneal infection presented with ascites. This case demonstrates the importance of diagnostic paracenteses, appropriate antibiotic therapy and the potential need to establish an alternative route for cerebrospinal fluid diversion in patients with ventriculoperitoneal shunts and ascites.

Bilirubin conjugate excretion and bilirubin uridine diphosphoglucuronyltransferase activity in nonjaundiced homozygous and heterozygous Gunn rats.

High-performance liquid chromatography was used to analyze the composition of bilirubin conjugates in bile from adult male and female outbred normal (JJ) and heterozygous (Jj) Gunn rats. Bile was examined directly without prior derivatization or extraction. Significantly higher bilirubin diglucuronide and lower bilirubin monoglucuronide (both C-8 and C-12 isomers) excretion in JJ rats was demonstrated. Bilirubin monoglucuronide C-8/C-12 ratios were similar in both genotypes, as was the percentage of bilirubin monoglucuronide monoglycoside diester. Hepatic bilirubin uridine diphosphoglucuronyltransferase activity showed a statistically significant positive correlation with the relative amount of bilirubin diglucuronide present in bile and a significant negative correlation with the amount of bilirubin monoglucuronide. The relative percentage of bilirubin monoglucuronide vs. diglucuronide in bile allows an indirect assessment of hepatic bilirubin uridine diphosphoglucuronyltransferase activity.