Search

At birth, human neonates are more likely to develop cholestasis and oxidative stress due to immaturity or other causes. We aimed to search for a potential association between bile acids profile, redox status, and type of diet in healthy infants.A cross-sectional, exploratory study enrolled 2-month-old full-term infants (n = 32). We measured plasma bile acids (total and conjugated), and red blood cell (RBC) oxidative stress biomarkers. The type of diet (breastfeeding, mixed, formula) was used as an independent variable.Plasma total bile acids medium value was 14.80 µmol/L (IQR: 9.25-18.00). The plasma-conjugated chenodeoxycholic acid percentage (CDCA%) correlated significantly and negatively with RBCs membrane-bound hemoglobin percentage (MBH%) (r = -0.635, p 0.01) and with RBC-oxidized glutathione (r = -0.403, p 0.05) levels. RBC oxidative stress biomarkers (especially MBH%) were predictors of conjugated CDCA%, and this predictive ability was enhanced when adjusted for the type of diet (MBH, r = 0.452, p 0.001).Our data suggest that the bile acid profile might play a role in the regulation of redox status (or vice versa) in early postnatal life. Eventually, the type of diet may have some impact on this process.The conjugated CDCA% in plasma is negatively correlated with biomarkers of RBC oxidative stress in healthy infants. Specific biomarkers of RBC oxidative stress (e.g. MBH, GSH, GSSG) may be promising predictors of conjugated CDCA% in plasma. The type of diet may influence the predictive ability of hit RBC oxidative stress biomarkers (e.g. MBH, GSH, GSSG). Our findings suggest a link between plasma bile acids profile and the RBC redox status in healthy infants, eventually modulated by the type of diet. The recognition of this link may contribute to the development of preventive and therapeutic strategies for neonatal cholestasis.

High levels of unconjugated bilirubin (UCB) can be neurotoxic. Nevertheless, the mechanism of UCB interaction with neural cells is still unknown. This study investigates whether cultured rat neurons and astrocytes respond differently to UCB exposure. UCB toxicity was evaluated by lactate dehydrogenase release, induction of apoptosis, cytoskeleton degeneration, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, and glutamate uptake. Primary cultures of rat brain astrocytes and neurons were incubated at 37 degrees C with 85.5 microM UCB plus 28.5 microM albumin for 4 h. In assays of glutamate uptake, cells were exposed to 80-120 microM UCB plus 100 microM albumin for 15 min. The results showed that after incubation with 85.5 microM UCB, lactate dehydrogenase release was greater in neurons than in astrocytes (38% versus 14%, p < 0.05). Also, levels of apoptosis were markedly enhanced in neurons (29% versus 19%, p < 0.01). In accordance, neuronal cytoskeleton disassembly was evident during incubation with 85.5 microM UCB, whereas equivalent effects on astrocytes required as much as 171 microM. Conversely, inhibition of MTT metabolism and glutamate uptake by UCB was more pronounced in astrocytes than in neurons (74% versus 60%, p < 0.05 and 41% to 56% versus 25% to 33%, p < 0.05, respectively). In conclusion, the study demonstrates that astrocytes are more susceptible to inhibition of glutamate uptake and MTT reduction by UCB, whereas neurons are more sensitive to cell death by necrosis or apoptosis. These results suggest that UCB is toxic to both astrocytes and neurons, although through distinct pathways.

The pathogenesis of bilirubin encephalopathy appears to result from accumulation of unconjugated bilirubin (UCB), which, in turn, may cause mitochondrial perturbation, release of intermembrane proteins, and, ultimately, cell death. Aging imparts to cells a different susceptibility to this toxic stimulus, as neonates are particularly vulnerable to the accumulation of UCB in the CNS. In this paper, we further characterize UCB-induced toxicity in isolated neuronal and glial cells according to age in culture. In addition, we investigate sensitivity of mitochondria derived from young and old rats to UCB-induced membrane permeabilization and, finally, evaluate whether age-dependent changes in UCB toxicity are accompanied by alterations in the mitochondrial content of cytochrome c. The results showed that UCB is more toxic to immature neural cells after 4-5 d in culture (p < 0.001), whereas neurons were more sensitive than astrocytes (p < 0.05). In fact, approximately 40% of cells were apoptotic in immature cultures compared with 20% in mature cultures. Unexpectedly, mitochondrial swelling and subsequent efflux of cytochrome c induced by UCB were 2-fold greater in organelles derived from older rats (p < 0.01). In conclusion, UCB toxicity of isolated rat neuronal and glial cells is modulated by age in culture in that immature cells are more susceptible. Mitochondria derived from younger rats are nevertheless more resistant to membrane permeabilization and cytochrome c release induced by UCB. The data indicate that the cells of young animals are relatively resistant to UCB toxicity, through a protective mechanism at the mitochondrial level; however, this is not sufficient to prevent apoptosis of cells in the young animal. Thus, although playing a role, direct mitochondrial injury may not be the sole mechanism of UCB cytotoxicity.

Elevated levels of free bilirubin in the brain triggered by increased concentrations of unconjugated bilirubin (UCB) may cause adverse neurological outcomes. We aimed to evaluate: (i)the effects of UCB in neuronal apoptosis, oxidative stress and P-JNK1/2 expression; (ii) if UCB damages the neuritic tree and whether glycoursodeoxycholic acid (GUDCA), IL-10 or L-NAME are protective; (iii)the brain regional susceptibility to UCB-induced dendritic disruption. Rat cortical neurons with 3 days in vitro (DIV) were incubated with 50 microM UCB plus 100 microM HSA. After 4h, we evaluated apoptosis (caspase-3 activity), P-JNK1/2 and nNOS expression (Western blot) and nitrites (Griess reagent). Following 24h, neurite arborization (MAP-2 labeling) was evaluated. Cells were also incubated with 50 microM GUDCA, 10 ng/mL IL-10 or 100 microM L-NAME prior to UCB exposure. Neurons from hippocampus and cerebellum at 3 DIV were also exposed to UCB (24h) to assess neuritic arborization. UCB increased caspase-3 activity (1.5-fold, p< 0.01), P-JNK1/2 (1.3-fold, p< 0.01) and nNOS (1.2- fold, p< 0.01), as well as NO production (1.7-fold, p< 0.01). Moreover, UCB decreased neurite extension (0.8-fold, p< 0.05) and ramification (0.5-fold, p< 0.05), effects that were prevented by GUDCA (p< 0.05 vs UCB), IL-10 and L-NAME (extension, p< 0.05; ramification, p< 0.01 vs UCB). Hippocampus revealed the utmost reduction in neurite ramification (46%, p< 0.01) and extension (28%, p< 0.05). Neuronal dysfunction by UCB comprises several intracellular pathways involving oxidative injury and disruption of neurite arborization. Anti-oxidant and/or anti-inflammatory compounds prevent UCB-damage to neuronal network. Hippocampus reveals the higher neurotoxicty by UCB. Funding:FCT- PTDC/SAU-NEU/64385/2006.