Standardization of phototherapy for neonatal hyperbilirubinemia using multiple-wavelength irradiance integration.

BACKGROUND: Phototherapy with radiation of 460-490 nm wavelengths provides the most potent therapeutic effect for neonatal jaundice. However, the efficacy of phototherapy has been estimated using single-wavelength detectors with sensitivity at approximately 460 nm. Cyclobilirubin formation capacity (CFC), which comprises the sum of the irradiance values from three wavelengths multiplied by their specific coefficients, has been proposed as an alternative marker to evaluate the efficacy of phototherapy. This study aimed to test whether two types of phototherapy devices with distinct spectral characteristics provide similar therapeutic effects on adjustment of device-to-patient distances to deliver similar CFCs. METHODS: Using a three-wavelength spectroradiometer, CFCs and footprints of the light-emitting diode and fluorescent tube devices were assessed. Having determined the device-specific distances that ensured similar CFCs, 32 newborn infants, requiring phototherapy for hyperbilirubinemia, were randomized into the light-emitting diode and fluorescent tube groups. The total serum bilirubin levels before and after phototherapy were assessed. RESULTS: The light-emitting diode and fluorescent tube devices had comparable CFCs at distances of 60 and 50 cm, respectively. Phototherapy reduced the total serum bilirubin levels from 18.1 to 14.6 mg/dL and from 19.1 to 15.1 mg/dL in the light-emitting diode and fluorescent tube groups, respectively. The two groups did not differ significantly with respect to the patients' clinical backgrounds, serum bilirubin levels, or changes before and after phototherapy. CONCLUSION: At similar CFCs, the two phototherapy devices reduced the total serum bilirubin levels by comparable amounts. Hence, determining CFCs may help predict phototherapy efficacy. This may ensure better safety and greater efficacy of the treatment for newborn infants.

Increased Carbon Monoxide Washout Rates in Newborn Infants.

BACKGROUND: Endogenous carbon monoxide (CO) production is primarily due to heme degradation, which also results in the equimolar production of bilirubin. Thus, estimates of total body CO production can serve as indices of total body bilirubin formation. The elimination rate of CO from a person's body (CO washout rate) after exposure to an elevated ambient CO concentration is determined by a variety of factors, and is very different between babies and adults. OBJECTIVE: We determined CO washout rates for babies using a simplified technique to measure total body CO excretion rates (VeCO). METHODS: Using a simplified technique, we measured the times to reach an approximate steady state after a change in ambient CO concentration (decay time constant) and CO washout rates in normal newborn infants using non-linear least squares curve fitting. RESULTS: We found a mean CO washout time of 18.7 ± 4.2 min and a CO equilibration (decay time) constant of 0.12 ± 0.04/min (0.08-0.21) for newborn infants. CONCLUSIONS: We confirm that CO washout rates for babies are much faster than those for adults. Therefore, measurements of carboxyhemoglobin (COHb) or end-tidal CO (ETCO), corrected for ambient CO, (COHbc and ETCOc, respectively) can be used as surrogates for VeCO and can provide accurate estimates of endogenous CO (VCO) and bilirubin production rates under normal environmental conditions. Such measurements can be used to identify infants with severe hyperbilirubinemia due to hemolysis and thus at high risk for bilirubin neurotoxicity.

Induction of Mild Hyperbilirubinemia: Hype or Real Therapeutic Opportunity?

Observational epidemiological studies showed that mild hyperbilirubinemia has beneficial effects on the prevention of cardiovascular disease, type 2 diabetes mellitus, and metabolic syndrome. In mammals, bilirubin plays a major role as a potent antioxidant. Uridine 5'-diphospho-glucuronosyl transferase (UGT)1A1 variants coding for bilirubin UDP-glucuronosyl transferase resulting in mild hyperbilirubinemia (as in Gilbert syndrome (GS)) may confer a strong genetic advantage. Strategies to boost bioavailability of bilirubin or to mimic GS represent an attractive approach to prevent many oxidative stress and inflammation-mediated diseases. Even a tiny, micromolar increase in serum bilirubin concentrations substantially decreases the risk of oxidative stress-mediated diseases. There are several possible ways to achieve this, including lifestyle changes, changes in dietary patterns, regular physical activities, or use of chemical drug or of specific plant products either in the form of regular food items or nutraceuticals. Further basic and experimental research is required to fully uncover this promising therapeutic field.

High unbound bilirubin for age: a neurotoxin with major effects on the developing brain.

Neonatal hyperbilirubinemia is one of the most frequent diagnoses made in neonates. A high level of unconjugated bilirubin that is unbound to albumin is neurotoxic when the level exceeds age-specific thresholds or at lower levels in neonates with neurotoxic risk factors. Lower range of unbound bilirubin results in apoptosis, while moderate-to-high levels result in neuronal necrosis. Basal ganglia and various brain stem nuclei are more susceptible to bilirubin toxicity. Proposed mechanisms of bilirubin-induced neurotoxicity include excessive release of glutamate, mitochondrial energy failure, release of proinflammatory cytokines, and increased intracellular calcium concentration. These mechanisms are similar to the events that occur following hypoxic-ischemic insult in neonates. Severe hyperbilirubinemia in term neonates has been shown to be associated with increased risk for autism spectrum disorders. The neuropathological finding of bilirubin-induced neurotoxicity also includes cerebellar injury with a decreased number of Purkinje cells, and disruption of multisensory feedback loop between cerebellum and cortical neurons which may explain the clinical characteristics of autism spectrum disorders. Severe hyperbilirubinemia occurs more frequently in infants from low- and middle-income countries (LMIC). Simple devices to measure bilirubin, and timely treatment are essential to reduce neurotoxicity, and improve outcomes for thousands of neonates around the world.

Cupping therapy: An analysis of the effects of suction on skin and the possible influence on human health.

BACKGROUND: Cupping therapy is a traditional therapy that has been employed worldwide for thousands of years. Despite a lack of quality clinical studies evaluating the efficacy of cupping therapy, its long history and widespread use throughout the world suggests the commonly claimed health benefits should not be completely discounted as without merit. PURPOSE: The goal of this paper is to present the research detailing what is known concerning the effects of suction on skin and underlying tissue, and the reaction of the body to that stimulus. Understanding the literature on the physiological effects of this mechanical force may help elaborate an explanation for the advertised local and systemic effects of cupping therapy. FINDINGS: Negative pressure causes stretching of the skin and underlying tissue and dilation of the capillaries. This stimulates an increase in tissue blood flow, eventually leading to capillary rupture and ecchymosis. Macrophages phagocytize the erythrocytes in the extravascular space which stimulates the production of Heme Oxygenase-1 (HO-1) to metabolize the heme. Heme catalysis results in the production of carbon monoxide (CO), biliverdin(BV)/bilirubin(BR) and iron. HO-1, BV, BR, and CO has been shown to have antioxidant, anti-inflammatory, antiproliferative, and neuromodulatory effects in animal and human systems. These substances also stimulate a shift of macrophages to the anti-inflammatory M2 phenotype. There is evidence that the effects are both local and systemic. CONCLUSION: Besides the mechanical effect of cupping increasing the local blood flow and stretching underlying tissue, activation of the HO-1 system could account for many of cupping therapy's claimed local and systemic health benefits.

Developmental, Genetic, Dietary, and Xenobiotic Influences on Neonatal Hyperbilirubinemia.

Hyperbilirubinemia, caused by the accumulation of unconjugated bilirubin, is one of the most common clinical diagnoses in both premature and term newborns. Owing to the fact that bilirubin is metabolized solely through glucuronidation by UDP-glucuronosyltransferase (UGT) 1A1, it is now known that immaturity of UGT1A1, in combination with the overproduction of bilirubin during the developmental stage, acts as a bottleneck to bilirubin elimination and predisposes the infant to high total serum bilirubin levels. Although neonatal jaundice is mostly benign, excessively high levels of serum bilirubin in a small percentage of newborns can cause bilirubin-induced neurologic dysfunction, potentially leading to permanent brain damage, a condition known as kernicterus Although a large portion of hyperbilirubinemia cases in newborns are associated with hemolytic diseases, we emphasize here the impaired ability of UGT1A1 to eliminate bilirubin that contributes to hyperbilirubinemia-induced neurotoxicity in the developmental stage. As a series of hereditary UGT1A1 mutations have been identified that are associated with UGT1A1 deficiency, new evidence has verified that delayed expression of UGT1A1 during the early stages of neonatal development is a tightly controlled event involving coordinated intrahepatic and extrahepatic regulation. This review recapitulates the progress that has been made in recent years in understanding the causes and physiopathology of severe hyperbilirubinemia, investigating molecular mechanisms underlying bilirubin-induced encephalopathy, and searching for potential therapies for treating pathologic hyperbilirubinemia. Several animal models have been developed to make it possible to examine bilirubin-induced neurotoxicity from multiple directions. Moreover, environmental factors that may alleviate or worsen the condition of hyperbilirubinemia are discussed.

Cadmium and arsenic override NF-κB developmental regulation of the intestinal UGT1A1 gene and control of hyperbilirubinemia.

Humanized UDP-glucuronosyltransferase (UGT)-1 (hUGT1) mice encode the UGT1 locus including the UGT1A1 gene. During neonatal development, delayed expression of the UGT1A1 gene leads to hyperbilirubinemia as determined by elevated levels of total serum bilirubin (TSB). We show in this report that the redox-sensitive NF-κB pathway is crucial for intestinal expression of the UGT1A1 gene and control of TSB levels. Targeted deletion of IKKß in intestinal epithelial cells (hUGT1/Ikkß(ΔIEC) mice) leads to greater neonatal accumulation of TSB than observed in control hUGT1/Ikkß(F/F) mice. The elevation in TSB levels in hUGT1/Ikkß(ΔIEC) mice correlates with a reduction in intestinal UGT1A1 expression. As TSB levels accumulate in hUGT1/Ikkß(ΔIEC) mice during the neonatal period, the increase over that observed in hUGT1/Ikkß(F/F) mice leads to weight loss, seizures and eventually death. Bilirubin accumulates in brain tissue from hUGT1/Ikkß(ΔIEC) mice inducing an inflammatory state as shown by elevated TNFα, IL-1ß and IL-6, all of which can be prevented by neonatal induction of hepatic or intestinal UGT1A1 and lowering of TSB levels. Altering the redox state of the intestines by oral administration of cadmium or arsenic to neonatal hUGT1/Ikkß(F/F) and hUGT1/Ikkß(ΔIEC) mice leads to induction of UGT1A1 and a dramatic reduction in TSB levels. Microarray analysis following arsenic treatment confirms upregulation of oxidation-reduction processes and lipid metabolism, indicative of membrane repair or synthesis. Our findings indicate that the redox state in intestinal epithelial cells during development is important in maintaining UGT1A1 gene expression and control of TSB levels.

Sulforaphane Suppresses Hepatitis C Virus Replication by Up-Regulating Heme Oxygenase-1 Expression through PI3K/Nrf2 Pathway.

Hepatitis C virus (HCV) infection-induced oxidative stress is a major risk factor for the development of HCV-associated liver disease. Sulforaphane (SFN) is an antioxidant phytocompound that acts against cellular oxidative stress and tumorigenesis. However, there is little known about its anti-viral activity. In this study, we demonstrated that SFN significantly suppressed HCV protein and RNA levels in HCV replicon cells and infectious system, with an IC50 value of 5.7 ± 0.2 µM. Moreover, combination of SFN with anti-viral drugs displayed synergistic effects in the suppression of HCV replication. In addition, we found nuclear factor erythroid 2-related factor 2 (Nrf2)/HO-1 induction in response to SFN and determined the signaling pathways involved in this process, including inhibition of NS3 protease activity and induction of IFN response. In contrast, the anti-viral activities were attenuated by knockdown of HO-1 with specific inhibitor (SnPP) and shRNA, suggesting that anti-HCV activity of SFN is dependent on HO-1 expression. Otherwise, SFN stimulated the phosphorylation of phosphoinositide 3-kinase (PI3K) leading Nrf2-mediated HO-1 expression against HCV replication. Overall, our results indicated that HO-1 is essential in SFN-mediated anti-HCV activity and provide new insights in the molecular mechanism of SFN in HCV replication.

A novel and sensitive assay for heme oxygenase activity.

Heme oxygenase (HO) is a renoprotective protein in the microsome that degrades heme and produces biliverdin. Biliverdin is then reduced to a potent antioxidant bilirubin by biliverdin reductase in the cytosol. Because HO activity does not necessarily correlate with HO mRNA or protein levels, a reliable assay is needed to determine HO activity. Spectrophotometric measurement is tedious and requires a relatively large amount of kidney samples. Moreover, bilirubin is unstable and spontaneously oxidized to biliverdin in vitro. We developed a novel and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify biliverdin to measure HO activity in mice. Biliverdin and its internal standard, a deuterated biliverdin-d4, have MS/MS fragments with m/z transitions of 583 to 297 and 587 to 299, respectively. We prepared lysates of mouse kidneys, and added excess hemin, NADPH, and bilirubin oxidase to convert all bilirubin produced to biliverdin. After 30-min incubation at 37 or 4°C, the samples were analyzed by LC-MS/MS. The difference in the amount of biliverdin between the two temperatures is HO activity. Treating mice with cobalt protoporphyrin, which induces the expression of HO, increased HO activity as determined by biliverdin production. Measuring the production of biliverdin using LC-MS/MS is a more sensitive and specific way to determine HO activity than the spectrophotometric method and allows the detection of subtle changes in renal or other HO activity.

Bilirubin production and hour-specific bilirubin levels.

OBJECTIVE: We assessed the relative contributions of increased bilirubin production (indexed by end-tidal carbon monoxide (CO) concentrations, corrected for ambient CO (ETCOc)) to hour-specific total bilirubin (TB) levels in healthy late preterm and term newborns. STUDY DESIGN: Post hoc analyses of concurrent ETCOc and TB (at 30±6 h of age) and follow-up TB levels at age 96±12 h and up to 168 h after birth were performed in a cohort of 641 term and late preterm infants. RESULTS: Increased bilirubin production (hour-specific ETCOc ⩾1.7 p.p.m. at age 30±6 h) was noted in ~80%, 42% and 32% of infants in the high-, intermediate- and low-risk TB zones, respectively. One infant with TB <40th percentile and ETCOc <1.7 p.p.m. developed TB ⩾95th percentile at age 168 h, probably due to decreased bilirubin elimination. CONCLUSIONS: Infants in the high-risk quartile of the hour-specific bilirubin nomogram have a higher mean bilirubin production. Infants with TB levels ⩾95th percentile without increased bilirubin production have impaired bilirubin elimination.

Living donor liver transplantation using a left liver extended to right anterior sector.

In living donor liver transplantations, right liver grafts have been commonly used to meet the metabolic demands of the recipient. However, a small left remnant liver volume sometimes limits its use due to donor safety concerns. Here, we report an innovative living donor hepatectomy using a left liver extended to the right anterior sector (segments 2-5 and 8), which can be considered for donors who are unsuited for right liver donation.

Hyperbilirubinemia, hemolysis, and increased bilirubin neurotoxicity.

Increased hemolysis in the presence of severe neonatal hyperbilirubinemia appears to augment the risk of bilirubin neurotoxicity. The mechanism of this intensifying effect is uncertain. In direct antiglobulin titer (DAT) positive, isoimmune hemolytic disease, the bilirubin threshold at which neurotoxicity occurs appears to be lower than in DAT-negative hyperbilirubinemia. In other hemolytic conditions, the hemolysis may simply facilitate the development of extremely high serum bilirubin levels. Whether the hemolytic process per se exerts an independent effect or whether a very rapid rise in serum bilirubin might lead to greater penetration of the blood-brain barrier is unclear. In this review, we survey the synergistic role of hemolysis associated with severe hyperbilirubinemia in the potentiation of bilirubin-induced neurotoxicity and suggest methods of identifying at-risk babies with increased hemolysis to allow for their increased surveillance.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) increases bilirubin formation but hampers quantitative hepatic conversion of biliverdin to bilirubin in rats with wild-type AH receptor.

In haem degradation, haem oxygenase-1 (HO-1) first cleaves haem to biliverdin, which is reduced to bilirubin by biliverdin IXα reductase (BVR-A). The environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes hepatic accumulation of biliverdin in moderately TCDD-resistant line B (Kuopio) rats. Using line B and two TCDD-sensitive rat strains, the present study set out to probe the dose-response and biochemical mechanisms of this accumulation. At 28 days after exposure to 3-300 µg/kg TCDD in line B rats, already the lowest dose of TCDD tested, 3 µg/kg, affected serum bilirubin conjugates, and after doses ≥100 µg/kg, the liver content of bilirubin, biliverdin and their conjugates (collectively 'bile pigments') as well as HO-1 was elevated. BVR-A activity and serum bile acids were increased only by the doses of 100 and 300 µg/kg TCDD, respectively. Biliverdin conjugates correlated best with biliverdin suggesting it to be their immediate precursor. TCDD (100 µg/kg, 10 days) increased hepatic bilirubin and biliverdin levels also in TCDD-sensitive Long-Evans (Turku/AB; L-E) rats. Hepatic bilirubin and bile acids, but not biliverdin, were increased in feed-restricted L-E control rats. In TCDD-sensitive line C (Kuopio) rats, 10 µg/kg of TCDD increased the body-weight-normalized biliary excretion of bilirubin. Altogether, the results suggest that at acutely toxic doses, TCDD induces the formation of bilirubin in rats. However, concurrently, TCDD seems to hamper the quantitative conversion of biliverdin to bilirubin in line B and L-E rats' liver. Biliverdin conjugates are most likely formed as secondary products of biliverdin.

William A Silverman lecture.

This is the text of the William A Silverman lecture given by Dr David K Stevenson at the Pediatric Academic Societies Annual Meeting in Washington, DC, May 4-7, 2013.

Development of an equation to correct for hemolysis in direct bilirubin measurements.

BACKGROUND: Direct bilirubin is measured for the investigation of pediatric and adult jaundice. Package inserts suggest that hemolysis decreases direct bilirubin measurements, but no published studies have adequately described the extent of interference. METHODS: The influence of hemolysis on direct bilirubin quantification (Beckman AU680) was evaluated by titrating increasing amounts of hemoglobin into specimens with variable starting concentrations of direct bilirubin. An equation was derived to predict the nominal interference-free concentration of direct bilirubin as a function of measured concentration and hemolysis-index. RESULTS: Hemolysis decreased the direct bilirubin concentration reported by the AU680. The extent of interference is a function of both the interference-free concentration of direct bilirubin and the degree of hemolysis. CONCLUSIONS: The concentration of direct bilirubin in hemolyzed specimens can be predicted.

Serum bilirubin may serve as a marker for increased heme oxygenase activity and inducibility in tissues--a rationale for the versatile health protection associated with elevated plasma bilirubin.

Unconjugated bilirubin functions intracellularly as a potent inhibitor of NADPH oxidase complexes, and albumin-bound bilirubin contributes significantly to the oxidant scavenging activity of plasma. So it is not surprising that serum levels of bilirubin have been found to correlate inversely with risk for vascular diseases and a host of other disorders. Nonetheless, recent Mendelian randomization analyses reveal that individuals who carry low expression alleles of the hepatic bilirubin conjugating enzyme UGT1A1, and hence have somewhat elevated levels of plasma bilirubin throughout life, are not at decreased risk for vascular disorders. This likely reflects the fact that, in most people, plasma levels of unconjugated, unbound bilirubin--the fraction of bilirubin capable of fluxing back into cells--are so low (near 1 nM) that they can exert only a trivial antioxidant influence on cells. In light of these findings, it is reasonable to propose that the inverse correlation of plasma bilirubin and disease risks noted in many studies often reflect the fact that elevated plasma bilirubin can serve as a marker for an increased propensity to generate bilirubin within cells. Consistent with this view, high expression alleles of the major enzymatic source of bilirubin, heme oxygenase-1 (HO-1), do associate with decreased vascular risk in the majority of studies that have addressed this issue, and increased plasma bilirubin has been reported in carriers of these alleles. Hence, the consistent reduction in vascular risk noted in people with Gilbert syndrome (traditionally defined as having serum bilirubin in excess of 20 µM) is likely attributable to an increased rate of bilirubin generation within tissues, rather than to the decreased hepatic UGT1A1 activity that characterizes this syndrome. However, there is good reason to suspect that, at some sufficiently high plasma bilirubin level--as in individuals with very intense Gilbert syndrome or in Gunn rats lacking UGT1A1 activity--the plasma bilirubin pool does indeed provide some antioxidant protection to cells. Strategies for boosting bilirubin production within cells via HO-1 induction, or for mimicking bilirubin's antioxidant activity with cyanobacterial phycobilins, may have important potential for health promotion.