Bill L. Kreamer

Use of a low-speed, iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations

SummaryA simple yet effective method (iso-density percoll centrifugation) has been developed for consistently preparing isolated rat liver parenchymal cells with over 98% initial viability. The method has been applied to cells isolated by a variety of collagenase digestion techniques. This procedure involves the low-speed centrifugation (50 ×g) of the initial cell suspension through a percoll medium having a density of 1.06 g/ml and results in the separation of single and viable parenchymal cells from cell aggregates, debris, and nonparenchymal cells. The enriched parenchymal cells have been shown to be superior to untreated cells by a number of criteria including: preparation homogeneity, cell morphology, maintenance of cytochrome P-450, hormonal responsiveness (measured by the induction of tyrosine aminotransferase after treatment with glucagon or dexamethasone, or both), plasma membrane integrity (determined by both trypan blue exclusion and leakage of glutamic-oxaloacetic transaminase), and the DNA repair capability after treatment with benzo[a]pyrene or 2-acetylaminofluorene.

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 μmol/L).

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 Fishers 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.

Embryotoxicity of 3,3′,4,4′-tetrachloroazobenzene and 3,3′,4,4′-tetrachioroazoxybenzene in the chick embryo

The toxicity of 3,3′,4,4′-tetrachloroazobenzene (TCAB) and 3,3′,4,4′-tetrachloroazoxybenzene (TCAOB) to chick embryos was examined. TCAB or TCAOB was dissolved in corn oil and injected into the air cell of fertile chicken eggs. The time of injection had a major effect on embryo mortality as eggs injected with TCAB or TCAOB on the fourth day of incubation had a higher incidence of embryo mortality than eggs injected on days 11–13. Both TCAB and TCAOB were more toxic than all other chemicals that have been tested in the chick embryo with the exception of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Comparing the potency of the two compounds, TCAOB was more potent than TCAB in the chick embryo with an estimated LD50 of 12 ng and 44 ng respectively. Rump edema was the major abnormality observed in embryos treated with either TCAB or TCAOB. Other malformations included altered feather pattern and lack of down, hemorrhage, external viscera, reduced body size, failure to withdraw the yolk sac, beak malformation, dilation of blood vessels, and monomicropthalmia. The results of this investigation suggest that both TCAB and TCAOB are teratogenic in the chick embryo.

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.

A controlled, randomized, double-blind trial of prophylaxis against jaundice among breastfed newborns.

Objectives. Neonatal jaundice is a greater problem for infants fed breast milk, compared with formula. This study tested the hypotheses that feeding breastfed newborns β-glucuronidase inhibitors during the first week after birth would increase fecal bilirubin excretion and would reduce jaundice without affecting breastfeeding deleteriously. Methods. Sixty-four breastfed newborns were randomized to 4 groups, ie, control or receiving 6 doses per day (5 mL per dose) of l-aspartic acid, enzymatically hydrolyzed casein (EHC), or whey/casein (W/C) for the first week. l-Aspartic acid and EHC inhibit β-glucuronidase. Transcutaneous bilirubin levels (primary outcome) were measured daily (Jaundice Meter [Minolta/Air Shields, Hatboro, PA] and Bilicheck [Respironics, Pittsburgh, PA]). All stools were collected, and fecal bile pigments, including bilirubin diglucuronide, bilirubin monoglucuronides, and bilirubin, were analyzed with high-performance liquid chromatography. Follow-up assessments included day 7 body weight, day 6/7 prebreastfeeding/postbreastfeeding weights, maternal ratings, and ages at formula introduction and breastfeeding cessation. Results. The groups were comparable at entry. Overall, the l-aspartic acid, EHC, and W/C groups had significantly lower transcutaneous bilirubin levels than did the control group (75.8%, 69.6%, and 69.2%, respectively, of the control mean, 8.53 mg/dL, at the bilirubin peak on day 4). The l-aspartic acid, EHC, and W/C groups had significantly lower transcutaneous bilirubin levels on days 3 to 7. Fecal bile pigment excretion was greatest in the l-aspartic acid group, significantly greater than control values. There were no significant differences in dosages, follow-up measurements, and maternal ratings. Conclusions. Use of minimal aliquots of l-aspartic acid and EHC for β-glucuronidase inhibition results in increased fecal bilirubin excretion and less jaundice, without disruption of the breastfeeding experience. Decreased jaundice in the W/C group, which lacked a β-glucuronidase inhibitor, suggests a different mechanism.

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.

Inhibition of β-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 β-glucuronidase is related to neonatal jaundice, this study examined the effect of a casein hydrolysate formula on β-glucuronidase.Methods:

A Novel Inhibitor of β-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 β-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 β-glucuronidase inhibition in hydrolyzed casein. A β-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 β-glucuronidase. The l-aspartic acid in hydrolyzed casein accounts for the majority of the β-glucuronidase inhibition present. Kinetic studies indicate a competitive inhibition mechanism. l-Aspartic acid is a newly identified competitive inhibitor of β-glucuronidase.

Measurement of Serum Bilirubin in Newborn Infants: Common Clinical Laboratory Methods versus High Performance Liquid Chromatography (HPLC)† 1522

Measurement of Serum Bilirubin in Newborn Infants: Common Clinical Laboratory Methods versus High Performance Liquid Chromatography (HPLC)† 1522