Screening methods for neonatal hyperbilirubinemia:benefits, limitations, requirements, and novel developments

Severe neonatal hyperbilirubinemia (SNH) is a serious condition that occurs worldwide. Timely recognition with bilirubin determination is key in the management of SNH. Visual assessment of jaundice is unreliable. Fortunately, transcutaneous bilirubin measurement for screening newborn infants is routinely available in many hospitals and outpatient settings. Despite a few limitations, the use of transcutaneous devices facilitates early recognition and appropriate management of neonatal jaundice. Unfortunately, however, advanced and often costly screening modalities are not accessible to everyone, while there is an urgent need for inexpensive yet accurate instruments to assess total serum bilirubin (TSB). In the near future, novel icterometers, and in particular optical bilirubin estimates obtained with a smartphone camera and processed with a smartphone application (app), seem promising methods for screening for SNH. If proven reliable, these methods may empower outpatient health workers as well as parents at home to detect jaundice using a simple portable device. Successful implementation of ubiquitous bilirubin screening may contribute substantially to the reduction of the worldwide burden of SNH. The benefits of non-invasive bilirubin screening notwithstanding, any bilirubin determination obtained through non-invasive screening must be confirmed by a diagnostic method before treatment. ImpactKey message: Screening methods for neonatal hyperbilirubinemia facilitate early recognition and timely treatment of severe neonatal hyperbilirubinemia (SNH). Any bilirubin screening result obtained must be confirmed by a diagnostic method. What does this article add to the existing literature? Data on optical bilirubin estimation are summarized. Niche research strategies for prevention of SNH are presented. Impact: Transcutaneous screening for neonatal hyperbilirubinemia contributes to the prevention of SNH. A smartphone application with optical bilirubin estimation seems a promising low-cost screening method, especially in low-resource settings or at home.Afdeling Klinische Chemie en Laboratoriumgeneeskunde (AKCL

Diagnostic methods for neonatal hyperbilirubinemia:benefits, limitations, requirements, and novel developments

Invasive bilirubin measurements remain the gold standard for the diagnosis and treatment of infants with severe neonatal hyperbilirubinemia. The present paper describes different methods currently available to assess hyperbilirubinemia in newborn infants. Novel point-of-care bilirubin measurement methods, such as the BiliSpec and the Bilistick, would benefit many newborn infants, especially in low-income and middle-income countries where the access to costly multi-analyzer in vitro diagnostic instruments is limited. Total serum bilirubin test results should be accurate within permissible limits of measurement uncertainty to be fit for clinical purposes. This implies correct implementation of internationally endorsed reference measurement systems as well as participation in external quality assessment programs. Novel analytic methods may, apart from bilirubin, include the determination of bilirubin photoisomers and bilirubin oxidation products in blood and even in other biological matrices. ImpactKey message: Bilirubin measurements in blood remain the gold standard for diagnosis and treatment of severe neonatal hyperbilirubinemia (SNH). External quality assessment (EQA) plays an important role in revealing inaccuracies in diagnostic bilirubin measurements. What does this article add to the existing literature? We provide analytic performance data on total serum bilirubin (TSB) as measured during recent EQA surveys. We review novel diagnostic point-of-care (POC) bilirubin measurement methods and analytic methods for determining bilirubin levels in biological matrices other than blood. Impact: Manufacturers should make TSB test results traceable to the internationally endorsed total bilirubin reference measurement system and should ensure permissible limits of measurement uncertainty.Afdeling Klinische Chemie en Laboratoriumgeneeskunde (AKCL

Functional Induction of the Cystine-Glutamate Exchanger System Xc- Activity in SH-SY5Y Cells by Unconjugated Bilirubin

We have previously reported that exposure of SH-SY5Y neuroblastoma cells to unconjugated bilirubin (UCB) resulted in a marked up-regulation of the mRNA encoding for the Na+ -independent cystine∶glutamate exchanger System Xc− (SLC7A11 and SLC3A2 genes). In this study we demonstrate that SH-SY5Y cells treated with UCB showed a higher cystine uptake due to a significant and specific increase in the activity of System Xc−, without the contribution of the others two cystine transporters (XAG− and GGT) reported in neurons. The total intracellular glutathione content was 2 folds higher in the cells exposed to bilirubin as compared to controls, suggesting that the internalized cystine is used for gluthathione synthesis. Interestingly, these cells were significantly less sensitive to an oxidative insult induced by hydrogen peroxide. If System Xc− is silenced the protection is lost. In conclusion, these results suggest that bilirubin can modulate the gluthathione levels in neuroblastoma cells through the induction of the System Xc−, and this renders the cell less prone to oxidative damage

Determinants of bilirubin neurotoxicity by an in vitro molecular approach

2010/2011Unconjugated Bilirubin (UCB) is the final product of the heme catabolism. The high serum UCB concentrations in the first days of life of the newborns, due to immature mechanisms for hepatic uptake, conjugation and biliary secretion, is called physiological neonatal jaundice. This common condition is generally a benign and transient phenomenon, but in some cases the hyperbilirubinemia can progress to bilirubin encephalopaties ranging from minimally neurological injury to severe and permanent neurodevelopmental dysfunction. In the present thesis the SH-SY5Y neuroblastoma cell line was used to approach the molecular events associated to bilirubin neurotoxicity and highlight the biochemical and molecular events that are induces in the neurons when get contact with the UCB. Depending on the bilirubin concentration and the time of exposure to UCB, we were able to define experimental setups for the study of bilirubin resistance and bilirubin toxicity. Using the model to study bilirubin resistance, it was demonstrated that the resistance is not entirely achieved by limiting the entrance or increasing extrusion of the pigment from the cell, but rather by enhancing the cellular defensive mechanisms, in particular against the oxidative stress. This was achieved by increasing the intracellular glutathione content via the specific induction of the genes and activity of the System Xc-. Furthermore, the cells exposed to bilirubin over-expressed several additional genes that encode for important antioxidant and detoxifying proteins like Heme Oxygenase-1 and NAD(P)H:quinone oxidoreductase 1. As far as the mechanisms of bilirubin neurotoxicity, we showed that UCB exposure lead to the induction of the intracellular ROS accumulation. Moreover, the data presented report evidences that the bilirubin toxicity could be displayed by a mechanism of excitotoxicity carried out by the cellular release of glutamate. Further studies will be necessary to elucidate the molecular mechanisms by which bilirubin produces neurotoxicity and to understand how the cells avoid the damage. The information presented here could contribute to the identification of targets to avoid the bilirubin damage.La bilirubina non coniugata (UCB) è il prodotto finale del catabolismo dell’eme. L’ alta concentrazione di bilirubina nei primi giorni di vita del neonato è conosciuto come ittero neonatale. Questa condizione è generalmente benigna e transitoria, ma in alcuni casi l’iperbilirubinemia può portare a encefalopatie che vanno da minimi danni neurologici a severe e permanenti disfunzioni neuronali. Nella presente tesi la linea cellulare di neuroblastoma umano SH-SY5Y è stata utilizzata per identificare gli eventi molecolari associati alla neurotossicità della bilirubina ed evidenziare gli eventi biochimici e molecolari che sono indotti nelle cellule neuronali quando queste sono esposte alla UCB. A seconda della concentrazione e del tempo di esposizione all’ UCB che viene utilizzato, si sono definiti due modelli sperimentali per studiare sia la resistenza delle cellule alla bilirubina che la sua tossicità. Utilizzando il modello per studiare la resistenza alla bilirubina, è stato dimostrato che la resistenza non è completamente dovuta alla limitazione dell’ entrata o alla rapida eliminazione del pigmento dalla cellula, ma da un aumento dei meccanismi di difesa cellulare, in particolare quelli contro lo stress ossidativo. Si è dimostrato che questo’ultimo è dovuto all’aumento intracellulare di glutatione attraverso l’induzione specifica di alcuni geni e ad un aumento dell’attività del Sistema Xc-. Inoltre, le cellule esposte a bilirubina overesprimono vari geni addizionali che codificano per importanti proteine antiossidanti e detossificanti come l’emossigenasi 1 e la NAD(P)H quinone ossidoreduttasi. Per quanto riguarda ai meccanismi di neurotossicità della bilirubina, è stato dimostrato che l’ esposizione a UCB induce l’accumulo intracellulare di ROS. Inoltre, i dati presentati evidenziano che la tossicità della bilirubina può essere dovuta a meccanismi di esototossicità prodotti dalla liberazione cellulare di glutammato. Ulteriori studi servirebbero per completare l’ identificazione dei meccanismi molecolari cui attraverso i quali la bilirubina produce il danno neuronale e capire come le cellule riescono a evitare il danno. L’informazione presentata in questo lavoro potrebbe contribuire all’identificazione di possibili targets che potrebbero prevenire il danno indotto da bilirubina.XXIV Ciclo198

Response to H₂O₂ oxidative stress of SH-SY5Y cells pretreated with bilirubin and GSH intracellular levels of the cells before H₂O₂ treatment.

<p>SH-SY5Y cells were exposed to two successive treatments: the first with 140 nM Bf (control with 0.6% DMSO medium) for 24 h. After this treatment, cells were released in growth medium for 156 h. Immediately after exposure to UCB (column A) and 156 h (column B) of release, cells were incubated, as second treatment, with different concentrations of H₂O₂ for 60 min and viability evaluated by MTT. At each time viability is expressed as a percentage of the controls considered as 100%. At each time viability results are compared with GSH intracellular levels of the cells before H₂O₂ treatment. Results are expressed as media ± SD of four different experiments.</p

Response to H₂O₂ oxidative stress in xCT silenced SH-SY5Y cells pre-treated with bilirubin.

<p><u>Panel A</u>: Protein expression of xCT (55 kDa and 35 kDa) was analyzed by Western blot after siRNA and Bf treatment. Lane 1: MOCK and 0.6% DMSO; lane 2: NT and 0.6% DMSO; lane 3: anti-xCT and DMSO 0.6%. Lane 4: MOCK and Bf 140 nM; lane 5: NT and Bf 140 nM ; lane 6: anti-xCT and Bf 140 nM. <u>Panel B</u>: Quantification of bands shown in lanes from 1 to 6 of panel A. xCT 35 kDa and 55 kDa bands normalized by actin and expressed as relative to MOCK . <u>Panel C</u>) H₂O₂ dose-response by MTT test was evaluated in untransfected (MOCK – left), transfected with not targeting siRNA (NT – middle) and transfected with siRNA against SLC7A11 gene (anti-xCT - right) SH-SY5Y cells. After silencing, and before 1 h H₂O₂ treatment, SH-SY5Y cells were exposed for 24 h to 0.6% DMSO or Bf 140 nM as indicated in the legend of each picture.</p

Sodium –dependent and independent L-[¹⁴C] cystine transport in SH-SY5Y cells.

<p>Transport activity was measured in control (0.6% DMSO, 24 h) and treated (Bf 140 nM, 24 h) cells. L-[¹⁴C] cystine (0.8 µM) uptake was measured after 10 min incubation at 37°C in the presence and absence of sodium ions. L-quisqualate (500 µM) was used as a specific inhibitor for System X_c⁻. Each point represents the mean ± SD of three plates of cells (** p<0.001).</p

Effect of bilirubin on mRNA expression levels of genes involved in cystine uptake.

<p>SH-SY5Y cells exposed to free medium (untreated), 0.6% DMSO medium or 140 nM Bf medium were collected after 1, 4 and 24 h of treatment. Specific mRNA expressions was analysed by qPCR. mRNA expression is shown as mean ± SD of 3 experiments relative to 1 h untreated control set at 1.0. **p<0.001 and *p<0.01 versus DMSO or untreated controls.</p

List of primer sequences designed for the quantification of specific mRNAs by qPCR.

<p>List of primer sequences designed for the quantification of specific mRNAs by qPCR.</p