Unbound bilirubin levels in phototherapy-treated preterm infants receiving soy-based lipid emulsion.

BACKGROUND: Phototherapy is an effective treatment for neonatal jaundice. Treatment indication uses total serum bilirubin (TSB), although unbound bilirubin (Bf) more accurately predicts disability risk. The goals of this investigation were to examine the response of Bf and TSB to phototherapy in preterm infants, and we hypothesized that (i) TSB and Bf respond differently; (ii) the relationship between TSB and Bf is altered; and (iii) unexpected Bf elevations are found. METHODS: One hundred and seventeen preterm infants <2 kg at birth and receiving (IL) were enrolled; and measurements of TSB and Bf were obtained. TSB was measured by the diazo method and Bf with a fluorescent Bf sensor BL22P1B11-Rh. RESULTS: Initial mean (± SD) TSB and Bf levels (41.4 ± 6.9 h) were 8.0 ± 9.0 mg/dL and 16.9 ± 12.4 nmol/L (P < 0.05). The rates of rise (ROR) were 0.21 ± 0.10 mg/dL/h for TSB and 0.38 ± 0.33 nmol/L/h for Bf. Phototherapy reduced TSB from 8.0 ± 9.0 to 5.8 ± 9.4 mg/dL (P = 0.068) but Bf did not change (16.9 ± 12.4 to 14.1 ± 9.4 nmol/L P = n.s.). Bf levels were >11 nmol/L in 64, >17 nmol/L in 18, and >22 nmol/L in 7 infants. CONCLUSIONS: Bf and TSB responded differently. While TSB and Bf correlated well before phototherapy, they did not correlate during phototherapy. TSB showed a trend toward a reduction with treatment, Bf did not. While TSB ROR information is not helpful, ROR Bf data can be utilized to anticipate treatment. Potentially high Bf levels existed before and after phototherapy and the mean Bf level at phototherapy termination remained elevated in a significant proportion of infants.

Interindividual Regulation of the Breast Cancer Resistance Protein/ABCG2 Transporter in Term Human Placentas.

The breast cancer resistance protein (BCRP/ABCG2) is a maternally-facing efflux transporter that regulates the placental disposition of chemicals. Transcription factors and gene variants are important regulatory factors that influence transporter expression. In this study, we sought to identify the genetic and transcriptional mechanisms underlying the interindividual expression of BCRP mRNA and protein across 137 term placentas from uncomplicated pregnancies. Placental expression of BCRP and regulatory transcription factor mRNAs was measured using multiplex-branched DNA analysis. BCRP expression and ABCG2 genotypes were determined using Western blot and Fluidigm Biomark genetic analysis, respectively. Placentas were obtained from a racially and ethnically diverse population, including Caucasian (33%), African American (14%), Asian (14%), Hispanic (15%), and mixed (16%) backgrounds, as well as unknown origins (7%). Between placentas, BCRP mRNA and protein varied up to 47-fold and 14-fold, respectively. In particular, BCRP mRNA correlated significantly with known transcription factor mRNAs, including nuclear factor erythroid 2-related factor 2 and aryl hydrocarbon receptor. Somewhat surprisingly, single-nucleotide polymorphisms (SNPs) in the ABCG2 noncoding regions were not associated with variation in placental BCRP mRNA or protein. Instead, the coding region polymorphism (C421A/Q141K) corresponded with 40%-50% lower BCRP protein in 421C/A and 421A/A placentas compared with wild types (421C/C). Although BCRP protein and mRNA expression weakly correlated (r = 0.25, P = 0.040), this relationship was absent in individuals expressing the C421A variant allele. Study results contribute to our understanding of the interindividual regulation of BCRP expression in term placentas and may help to identify infants at risk for increased fetal exposure to chemicals due to low expression of this efflux protein.

Effects of Soybean Lipid Infusion on Unbound Free Fatty Acids and Unbound Bilirubin in Preterm Infants.

OBJECTIVE: To assess the effects of a soybean lipid emulsion infusions on levels of unbound (free) bilirubin (Bf) and unbound free fatty acids (FFAu) as well as changes in Bf and total serum bilirubin (TSB) during phototherapy in infants born preterm. STUDY DESIGN: Ninety-seven infants born preterm (birth weight: 500-2000 g; gestational age: 23-34 weeks) were enrolled to investigate the effect of 0, 1, 2, and 3 g/kg/d of intralipid infusion on Bf and FFAu. Pre- and postphototherapy TSB, FFAu, and Bf also were analyzed in 91 infants to assess the effects of phototherapy. FFAu levels were measured with the fluorescent probe ADIFAB2 and Bf by the fluorescent Bf sensor BL22P1B11-Rh during intralipid infusion and at start and end of phototherapy. TSB and plasma albumin were measured by the diazo and bromcresol green techniques, respectively. Bilirubin-albumin dissociation constants were calculated based on Bf and plasma albumin. RESULTS: Bf and FFAu increased with increasing intralipid dosage across all gestational ages. TSB and Bf were correlated significantly when infants received 0 or 1 g/kg/d of intralipid but not at greater doses of intralipid (2 and 3 g/kg/d). Although phototherapy effectively reduced both TSB and Bf in the total phototherapy group (by 32% and 12%, respectively), it reduced TSB, but not Bf, in infants less than 28 weeks of gestation. CONCLUSIONS: Increasing intralipid doses result in increasing FFAu levels, which are associated with increased Bf independent of TSB. In infants born extremely preterm (<28 weeks of gestation), phototherapy effectively reduces TSB but not Bf.

Dysregulation of bile acid homeostasis in parenteral nutrition mouse model.

Long-term parenteral nutrition (PN) administration can lead to PN-associated liver diseases (PNALD). Although multiple risk factors have been identified for PNALD, to date, the roles of bile acids (BAs) and the pathways involved in BA homeostasis in the development and progression of PNALD are still unclear. We have established a mouse PN model with IV infusion of PN solution containing soybean oil-based lipid emulsion (SOLE). Our results showed that PN altered the expression of genes involved in a variety of liver functions at the mRNA levels. PN increased liver gene expression of Cyp7a1 and markedly decreased that of Cyp8b1, Cyp7b1, Bsep, and Shp. CYP7A1 and CYP8B1 are important for synthesizing the total amount of BAs and regulating the hydrophobicity of BAs, respectively. Consistently, both the levels and the percentages of primary BAs as well as total non-12α-OH BAs increased significantly in the serum of PN mice compared with saline controls, whereas liver BA profiles were largely similar. The expression of several key liver-X receptor-α (LXRα) target genes involved in lipid synthesis was also increased in PN mouse livers. Retinoid acid-related orphan receptor-α (RORα) has been shown to induce the expression of Cyp8b1 and Cyp7b1, as well as to suppress LXRα function. Western blot showed significantly reduced nuclear migration of RORα protein in PN mouse livers. This study shows that continuous PN infusion with SOLE in mice leads to dysregulation of BA homeostasis. Alterations of liver RORα signaling in PN mice may be one of the mechanisms implicated in the pathogenesis of PNALD.

Inherited disorders of bilirubin clearance.

Inherited disorders of hyperbilirubinemia may be caused by increased bilirubin production or decreased bilirubin clearance. Reduced hepatic bilirubin clearance can be due to defective (i) unconjugated bilirubin uptake and intrahepatic storage, (ii) conjugation of glucuronic acid to bilirubin (e.g., Gilbert syndrome, Crigler-Najjar syndrome, Lucey-Driscoll syndrome, breast milk jaundice), (iii) bilirubin excretion into bile (Dubin-Johnson syndrome), or (iv) conjugated bilirubin re-uptake (Rotor syndrome). In this review, the molecular mechanisms and clinical manifestations of these conditions are described, as well as current approaches to diagnosis and therapy.

Suppression of Bile Acid Synthesis in a Preterm Infant Receiving Prolonged Parenteral Nutrition.

Bile acid metabolism is altered in neonates on parenteral nutrition (PN), predisposing them to parenteral nutrition-associated liver disease. Cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in the bile acid synthesis pathway, is repressed by fibroblast growth factor 19 (FGF19) and phytosterols (PS). We describe a case of a preterm infant who developed necrotizing enterocolitis (NEC) and received exclusive PN for over 2 months. Our objective was to serially assess CYP7A1 activity and plasma FGF19 and PS concentrations in this infant case compared to five healthy preterm infants. We found that CYP7A1 activity increased during the first 2 weeks of life in control infants but was undetectable in the infant case. FGF19 concentrations were high at birth in all infants and subsequently declined and did not differ between the case and control infants. As expected, PS concentrations were elevated in the infant case and continued to increase despite lipid minimization. In conclusion, CYP7A1 activity was gradually upregulated in healthy preterm infants but remained suppressed in the infant requiring prolonged PN. Preterm infants also had elevated FGF19 concentrations at birth, which decreased with advancing postnatal age.

Plasma unbound free fatty acid profiles in premature infants before and after intralipid infusion.

Background: Unbound free fatty acids (FFAu) are the bioactive fraction of plasma free fatty acids (FFA). Most plasma FFA are bound to albumin. Only when FFA dissociate from albumin, do they become biologically active.Objective: To measure the first FFAu profiles in human infants and to measure these profiles before and during intravenous administration of the soybean lipid, intralipid (IL).Study design: The study population was 16 premature infants, from a parent study of 130 infants with birth weights 500-2000 g and gestational age 23-34 weeks. The infants chosen had plasma samples of ≥120 µL (volume needed for each FFAu profile measurement) in the first day of life. Infants received IL infusions starting in the second day of life at 1 g/kg/day, increasing by 1-g/kg/day daily up to 3 g/kg/day. FFAu profiles were determined during IL infusion when plasma was available. Profiles are the concentrations of the nine most abundant long-chain FFAu and were determined using novel fluorescent probes.Results: Before intralipid infusion unbound myristic acid was the dominant FFAu, as high as 78% of the total FFAu (sum of the 9 FFAu). In contrast, unbound linoleic acid was 0% in all infants. With increasing infusion of IL to 3 g/kg/day, unbound linoleic increased to 26% of the total FFAu, with unbound oleic, myristic, and linolenic acid the second, third and fourth most abundant. The average total FFAu concentration also increased from 4 nM before intralipid to 53 nM at 3 g/kg/day. During IL infusion the FFAu profiles approached the fatty acid composition of intralipid at 3 g/kg/day.Conclusions: This first study of FFAu profiles in neonates revealed that before IL infusion unbound linoleic acid was zero in all 16 infants and levels of myristic acid were exceptionally large, as much as 78% of the total FFAu profile. These results suggest important and previously unrecognized roles of lipid metabolism in early development. Zero unbound linoleic acid before IL infusion may help promote closure of the ductus arteriosus but after IL infusion, synthesis of arachidonic from linoleic acid may tend to promote patency. The high levels of unbound myristate may be needed for immediate neonatal energy needs.

Developmental regulation of the gut-liver (FGF19-CYP7A1) axis in neonates.

Introduction: Fibroblast growth factor 19 (FGF19) is a gut-derived hormone that regulates the expression of CYP7A1, the rate-limiting enzyme in bile acid (BA) synthesis pathway. Dysregulation of the FGF19-CYP7A1 (gut-liver) axis is associated with cholestatic liver disease. Infants, especially preterm infants and those with intestinal failure are at high risk for developing cholestatic liver disease. The activity of the gut-liver axis has not been characterized in this population. Our objective was to assess relationships between circulating FGF19 concentrations and CYP7A1 activity in neonates.Materials and methods: Plasma samples were obtained longitudinally from term and preterm infants (22-41-week gestation) hospitalized in a neonatal intensive care unit. Infants with congenital and acquired gastrointestinal disorders were excluded. Plasma levels of 7α-hydroxy-4-cholesten-3-one (C4), a marker of CYP7A1 activity, were quantified using HPLC-MS/MS. Plasma FGF19 concentrations were quantified by ELISA. Data were analyzed using linear regression models and structural equation modeling.Results: One hundred eighty-one plasma samples were analyzed from 62 infants. C4 concentrations were undetectable prior to 30 weeks' gestation and, thereafter, increased with advancing gestational age and with volume of enteral feeds. They did not correlate with serum FGF19 concentrations, which decreased with advancing gestational age and volume of enteral feeds.Discussion: The activity of CYP7A1, the rate-limiting BA synthetic enzyme in adults, is developmentally regulated and undetectable in newborns less than 30 weeks' gestation. FGF19 concentrations do not correlate with CYP7A1 activity, suggesting that the gut-liver axis is not functional in infants. High FGF19 concentrations at birth in infants less than 37 weeks' gestation is a novel finding, and its source and role in preterm infants warrants further investigation.Rationale: The intestinal hormone, fibroblast growth factor 19 (FGF19), is a major regulator of CYP7A1, the rate limiting enzyme in bile acid (BA) synthesis. Recently, dysregulation of the gut-liver (FGF19-CYP7A1) axis has been implicated in adult cholestatic liver disease, and animal studies have shown that exogenous FGF19 protects against liver injury. Given the therapeutic potential related to this signaling pathway, we sought to characterize the association between CYP7A1 and FGF19 in term and preterm infants. We conducted a prospective, observational study that measured in vivo CYP7A1 activity and FGF19 concentrations in 62 term and preterm infants (n = 181 samples). We found that CYP7A1 activity is developmentally regulated; its activity is undetectable prior to 30 weeks' gestation and increases with advancing gestational age and volume of enteral feeds. Contrary to expectation, we demonstrated that FGF19 is expressed at birth in preterm infants and decreases over time, even as enteral feeds increase. Using structural equation modeling, we were able to show that CYP7A1 activity does not correlate with FGF19 concentrations. Our results suggest that the gut-liver axis is not upregulated in preterm and term infants and that neonates with cholestatic liver disease will unlikely benefit from supplemental FGF19. We also report novel findings of elevated FGF19 concentrations in preterm infants at birth and speculate that there may be an extra-intestinal source of FGF19 that is developmentally expressed in these infants.

Effects of total parenteral nutrition on drug metabolism gene expression in mice.

Parenteral nutrition-associated liver disease (PNALD) is a liver dysfunction caused by various risk factors presented in patients receiving total parenteral nutrition (TPN). Omega-6 rich Intralipid® and omega-3 rich Omegaven® are two intravenous lipid emulsions used in TPN. TPN could affect the hepatic expression of genes in anti-oxidative stress, but it's unknown whether TPN affects genes in drug metabolism. In this study, either Intralipid®- or Omegaven®-based TPN was administered to mice and the expression of a cohort of genes involved in anti-oxidative stress or drug metabolism was analyzed, glutathione (GSH) levels were measured, and protein levels for two key drug metabolism genes were determined. Overall, the expression of most genes was downregulated by Intralipid®-based TPN (Gstp1, Gstm1, 3, 6, Nqo1, Ho-1, Mt-1, Gclc, Gclm, Cyp2d9, 2f2, 2b10, and 3a11). Omegaven® showed similar results as Intralipid® except for preserving the expression of Gstm1 and Cyp3a11, and increasing Ho-1. Total GSH levels were decreased by Intralipid®, but increased by Omegaven®. CYP3A11 protein levels were increased by Omegaven®. In conclusion, TPN reduced the expression of many genes involved in anti-oxidative stress and drug metabolism in mice. However, Omegaven® preserved expression of Cyp3a11, suggesting another beneficial effect of Omegaven® in protecting liver functions.

Efficacy, safety, and tolerability of the low glycemic index treatment in pediatric epilepsy.

PURPOSE: To report the efficacy, safety, and tolerability of the low glycemic index treatment (LGIT) in pediatric epilepsy. METHODS: A retrospective chart review was performed on patients initiating the LGIT at the Massachusetts General Hospital between January 2002 and June 2008. Demographic and clinical information including seizure type, baseline seizure frequency, medications, blood chemistries, side effects, and anthropometrics were collected. Initiation of the LGIT was done in an outpatient setting. Patients were educated by a dietitian to restrict foods with high glycemic index and to limit total daily carbohydrates to 40-60 g. Change in seizure frequency was assessed at 1-, 3-, 6-, 9-, and 12-month follow-up intervals. RESULTS: Seventy-six children were included in the study. Eighty-nine percent had intractable epilepsy (>or=3 antiepileptic drugs). A greater than 50% reduction from baseline seizure frequency was observed in 42%, 50%, 54%, 64%, and 66% of the population with follow-up available at 1, 3, 6, 9, and 12 months, respectively. Increased efficacy was correlated with lower serum glucose levels at some time points, but not with beta-hydroxybutyrate (BOHB) changes or ketosis status at any time point. Only three patients reported side effects (transient lethargy). Blood urea nitrogen (BUN) was elevated in approximately one-third of follow-up laboratory studies. No significant changes were seen in body mass index (BMI) or BMI z-score at any follow-up interval. The most cited reason for treatment discontinuation was the restrictiveness of the diet, in 18 patients (24%). CONCLUSION: The LGIT was associated with reduced seizure frequency in a large fraction of patients, with limited side effects.

Essential Fatty Acid Deficiency with SMOFlipid Reduction in an Infant with Intestinal Failure-Associated Liver Disease.

Multicomponent lipid emulsions, such as SMOFlipid, contain intermediate amounts of essential fatty acids (EFAs) compared with traditional soybean-oil based lipid emulsions and 100% fish-oil lipid emulsions. We describe the development of moderate EFA deficiency (EFAD) and slow weight gain in an infant with intestinal failure-associated liver disease managed with SMOFlipid reduction (1 g/kg/d). Once SMOFlipid dosage was increased (2-3 g/kg/d), EFA levels normalized, adequate growth resumed, and the infant's cholestasis resolved. We recommend avoiding lipid reduction of SMOFlipid, which not only increases the risk for EFAD, but also is unnecessary given that cholestasis can be reversed on conventional doses of SMOFlipid.

Unbound bilirubin measurements by a novel probe in preterm infants.

BACKGROUND: Hyperbilirubinemia occurs in over 80% of newborns and severe bilirubin toxicity can lead to neurological dysfunction and death, especially in preterm infants. Currently, the risk of bilirubin toxicity is assessed by measuring the levels of total serum bilirubin (TSB), which are used to direct treatments including immunoglobulin administration, phototherapy, and exchange transfusion. However, free, unbound bilirubin levels (Bf) predict the risk of bilirubin neurotoxicity more accurately than TSB. OBJECTIVE: To examine Bf levels in preterm infants and determine the frequency with which they exceed reported neurotoxic thresholds. METHODS: One hundred thirty preterm infants (BW 500-2000 g; GA 23-34 weeks) were enrolled and Bf levels measured during the first week of life by the fluorescent Bf sensor BL22P1B11-Rh. TSB and plasma albumin were measured by standard techniques. Bilirubin-albumin dissociation constants (Kd) were calculated based on Bf and plasma albumin. RESULTS: Five hundred eighty samples were measured during the first week of life, with an overall mean Bf of 13.6 ± 9.0 nM. A substantial number of measurements exceeded potential toxic thresholds levels as reported in the literature. The correlation between Bf and TSB was statistically significant (r2 0.17), but this weak relationship was lost at high Bf levels. Infants <28-week gestations had more hearing screening failures than infants ≥28-week gestation. CONCLUSIONS: Unbound (free) bilirubin values are extremely variable during the first week of life in preterm infants. A significant proportion of these values exceeded reported neurotoxic thresholds.

Effects of soybean lipid infusion on triglyceride and unbound free fatty acid levels in preterm infants.

Objective: To determine the plasma triglyceride (TG) and unbound free fatty acid (FFAu) levels in infants treated with increasing dosages of soybean lipid, intralipid (IL), infusion. Study design: TG and FFAu levels were measured in 78 preterm infants (BW 500-2000 g; GA 23-34 weeks) using the fluorescent probe ADIFAB2 and enzymatic method. Results: The infants' BW was 1266.2 ± 440.7 g and GA 28.8 ± 3.1 weeks. TG levels were 77.4 ± 50 mg/dL, 140.2 ± 188 mg/dL (p < .04 compared to levels during low dose IL infusion) and 135.6 ± 118 mg/dL (p < .004), respectively during increased IL rates. FFAu levels were 17.7 ± 13 nM, 47.3 ± 102.8 nM (p = .07) and 98 ± 234 nM (p = .03). TG levels correlated with IL dose, the rate of IL administration, and FFAu levels. TG and FFAu levels were higher in infants below 28 weeks' gestation Conclusions: Increasing dosage of IL is associated with increasing levels of TG and FFAu, especially in infants below 29 weeks of gestation. The increased level of FFAu suggests inefficient cellular utilization.

Regional expression of the BCRP/ABCG2 transporter in term human placentas.

The breast cancer resistance protein (BCRP, ABCG2) is an efflux transporter that removes xenobiotics that cross the placenta back to the maternal circulation, thereby limiting exposure of the fetus to drugs and chemicals. Currently, variability of BCRP expression within the placenta is not known. Ten placentas were collected from healthy women undergoing elective Cesarean sections at term. Villous samples were dissected in defined regions (medial, intermediate, and peripheral) and BCRP mRNA and protein were quantified. There were no regional differences in mRNA expression of housekeeping genes (GAPDH, RPL13a, PRL, 18S). GAPDH had the lowest correlation with BCRP Ct values and was used for BCRP mRNA normalization. No differences in placental BCRP mRNA and protein were observed among the sample sites (<20% variability). Sampling site does not affect the expression of BCRP, supporting the utility of single site sampling protocols to assess the interindividual regulation of this transporter in human placentas.