Olfactory Function is Affected in Patients with Cirrhosis Depending on the Severity of Hepatic Encephalopathy.

INTRODUCTION AND AIM: Olfactory functions are altered to a variable degree by chronic liver disease. Few studies including only small populations of patients emphasized the possibility of hepatic encephalopathy (HE) influencing olfactory nervous tasks. So far, no study has explicitly focused on olfactory function depending on the severity of HE as assessed by objective diagnostic procedures. Thus we performed a study using the "Sniffin' Sticks" test system, critical flicker-fusion frequency (CFF) and clinical West Haven criteria. MATERIAL AND METHODS: 54 cirrhotic patients with liver cirrhosis were included. Furthermore, 43 adult volunteers participating as a non-cirrhotic control group. Olfactory testing was performed using the "Sniffin' Stick" test battery (Burghart Medizintechnik, Wedel, Germany) which renders a widely-used tool both in clinical and research settings for the assessment of olfactory threshold, odor identification and discrimination. Several complications of cirrhosis were diagnosed by reference methods. Statistical analysis of cirrhosis-associated complications and their relation to olfactory function was performed. Assessment of HE and classification of different stages were performed according to clinical criteria (West- Haven criteria) and according to CFF, which was determined using a portable analyzer. RESULTS: Olfactory function was significantly reduced in cirrhotic patients (in 61.1%) compared to controls (p < 0.001). Among cirrhotics patients, the prevalence of olfactory deficits (hyposmia, anosmia) increased with the severity of HE as assessed by CFF and clinical criteria (p = 0.008 and p = 0.097, respectively). No correlation was observed between olfactory deficits and severity of liver disease as assessed by Child-Pugh-Score, etiology of cirrhosis and complications of cirrhosis such as ascites and portal venous hypertension. CONCLUSIONS: Olfactory testing serves as a screening tool for HE and may facilitate grading of HE-severity.

BDNF and PDE4, but not the GRPR, regulate viability of human medulloblastoma cells.

Medulloblastoma is the most common brain tumor of childhood. Emerging molecular targets in medulloblastoma include neurotrophin and neuropeptide receptors. In the present study, we have examined the influence of brain-derived neurotrophic factor (BDNF)/TrkB receptor- and gastrin-releasing peptide receptor (GRPR)-mediated signaling on the viability of human medulloblastoma cells. The expression of TrkB and GRPR was confirmed by immunohistochemistry and mRNA for both BDNF and GRPR was detected by reverse transcriptase polymerase chain reaction in Daoy, D283, and ONS76 cells. Treatment with BDNF significantly inhibited the viability of Daoy and D283, but not ONS76 cells, measured with the MTT assay. Neither the GRPR agonists GRP and bombesin nor the GRPR antagonist RC-3095 affected cell viability. Because previous findings have indicated that the viability of glioma cells might be enhanced by GRP when combined with the cAMP phosphodiesterase-4 (PDE4) inhibitor rolipram, we also examined the effects of rolipram alone or combined with GRP on cell viability. Rolipram significantly reduced the viability of all three cell lines, and the inhibitory effect of rolipram in Daoy cells was not modified by cotreatment with GRP. The results suggest that BDNF/TrkB and PDE4, but not the GRPR, regulate the viability of medulloblastoma cells.

Inhibition of brain energy metabolism by the branched-chain amino acids accumulating in maple syrup urine disease.

In the present work we investigated the in vitro effect of the branched-chain amino acids (BCAA) accumulating in maple syrup urine disease (MSUD) on some parameters of energy metabolism in cerebral cortex of rats. 14CO2 production from [1-14C]acetate, [1-5-14C]citrate and [U-14C]glucose, as well as glucose uptake by the brain were evaluated by incubating cortical prisms from 30-day-old rats in the absence (controls) or presence of leucine (Leu), valine (Val) or isoleucine (Ile). All amino acids significantly reduced 14CO2 production by around 20-55%, in contrast to glucose utilization, which was significantly increased by up to 90%. Furthermore, Leu significantly inhibited the activity of the respiratory chain complex IV, whereas Val and Ile markedly inhibited complexes II-III, III and IV by up to 40%. We also observed that trolox (alpha-tocopherol) and creatine totally prevented the inhibitory effects provoked by the BCAA on the respiratory chain complex activities, suggesting that free radicals were involved in these effects. The results indicate that the major metabolites accumulating in MSUD disturb brain aerobic metabolism by compromising the citric acid cycle and the electron flow through the respiratory chain. We presume that these findings may be of relevance to the understanding of the pathophysiology of the neurological dysfunction of MSUD patients.

Evidence that glutaric acid reduces glutamate uptake by cerebral cortex of infant rats.

The role of excitotoxicity in the cerebral damage of glutaryl-CoA dehydrogenase deficiency (GDD) is under intense debate. We therefore investigated the in vitro effect of glutaric (GA) and 3-hydroxyglutaric (3-OHGA) acids, which accumulate in GDD, on [(3)H]glutamate uptake by slices and synaptosomal preparations from cerebral cortex and striatum of rats aged 7, 15 and 30 days. Glutamate uptake was significantly decreased by high concentrations of GA in cortical slices of 7-day-old rats, but not in cerebral cortex from 15- and 30-day-old rats and in striatum from all studied ages. Furthermore, this effect was not due to cellular death and was prevented by N-acetylcysteine preadministration, suggesting the involvement of oxidative damage. In contrast, glutamate uptake by brain slices was not affected by 3-OHGA exposure. Immunoblot analysis revealed that GLAST transporters were more abundant in the cerebral cortex compared to the striatum of 7-day-old rats. Moreover, the simultaneous addition of GA and dihydrokainate (DHK), a specific inhibitor of GLT1, resulted in a significantly higher inhibition of [(3)H]glutamate uptake by cortical slices of 7-day-old rats than that induced by the sole presence of DHK. We also observed that both GA and 3-OHGA exposure did not alter the incorporation of glutamate into synaptosomal preparations from cerebral cortex and striatum of rats aged 7, 15 and 30 days. Finally, GA in vivo administration did not alter glutamate uptake into cortical slices from 7-day-old rats. Our findings may explain at least in part why cortical neurons are more vulnerable to damage at birth as evidenced by the frontotemporal cortical atrophy observed in newborns affected by GDD.

Age and brain structural related effects of glutaric and 3-hydroxyglutaric acids on glutamate binding to plasma membranes during rat brain development.

(1) In the present study we determined the effects of glutaric (GA, 0.01-1 mM) and 3-hydroxyglutaric (3-OHGA, 1.0-100 microM) acids, the major metabolites accumulating in glutaric acidemia type I (GA I), on Na(+)-independent and Na(+)-dependent [(3)H]glutamate binding to synaptic plasma membranes from cerebral cortex and striatum of rats aged 7, 15 and 60 days. (2) GA selectively inhibited Na(+)-independent [(3)H]glutamate binding (binding to receptors) in cerebral cortex and striatum of rats aged 7 and 15 days, but not aged 60 days. In contrast, GA did not alter Na(+)-dependent glutamate binding (binding to transporters) to synaptic membranes from brain structures of rats at all studied ages. Furthermore, experiments using the glutamatergic antagonist CNQX indicated that GA probably binds to non-NMDA receptors. In addition, GA markedly inhibited [(3)H]kainate binding to synaptic plasma membranes in cerebral cortex of 15-day-old rats, indicating that this effect was probably directed towards kainate receptors. On the other hand, experiments performed with 3-OHGA revealed that this organic acid did not change Na(+)-independent [(3)H]glutamate binding to synaptic membranes from cerebral cortex and striatum of rats from all ages, but inhibited Na(+)-dependent [(3)H]glutamate binding to membranes in striatum of 7-day-old rats, but not in striatum of 15- and 60-day-old rats and in cerebral cortex of rats from all studied ages. We also provided some evidence that 3-OHGA competes with the glutamate transporter inhibitor L-trans-pyrrolidine-2,4-dicarboxylate, suggesting a possible interaction of 3-OHGA with glutamate transporters on synaptic membranes. (3) These results indicate that glutamate binding to receptors and transporters can be inhibited by GA and 3-OHGA in cerebral cortex and striatum in a developmentally regulated manner. It is postulated that a disturbance of glutamatergic neurotransmission caused by the major metabolites accumulating in GA I at early development may possibly explain, at least in part, the window of vulnerability of striatum and cerebral cortex to injury in patients affected by this disorder.

Differential inhibitory effects of methylmalonic acid on respiratory chain complex activities in rat tissues.

Methylmalonic acidemia is an inherited metabolic disorder biochemically characterized by tissue accumulation of methylmalonic acid (MMA) and clinically by progressive neurological deterioration and kidney failure, whose pathophysiology is so far poorly established. Previous studies have shown that MMA inhibits complex II of the respiratory chain in rat cerebral cortex, although no inhibition of complexes I-V was found in bovine heart. Therefore, in the present study we investigated the in vitro effect of 2.5mM MMA on the activity of complexes I-III, II, II-III and IV in striatum, hippocampus, heart, liver and kidney homogenates from young rats. We observed that MMA caused a significant inhibition of complex II activity in striatum and hippocampus (15-20%) at low concentrations of succinate in the medium, but not in the peripheral tissues. We also verified that the inhibitory property of MMA only occurred after exposing brain homogenates for at least 10 min with the acid, suggesting that this inhibition was mediated by indirect mechanisms. Simultaneous preincubation with the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME) and catalase (CAT) plus superoxide dismutase (SOD) did not prevent MMA-induced inhibition of complex II, suggesting that common reactive oxygen (superoxide, hydrogen peroxide and hydroxyl radical) and nitric (nitric oxide) species were not involved in this effect. In addition, complex II-III (20-35%) was also inhibited by MMA in all tissues tested, and complex I-III only in the kidney (53%) and liver (38%). In contrast, complex IV activity was not changed by MMA in all tissues studied. These results indicate that MMA differentially affects the activity of the respiratory chain pending on the tissues studied, being striatum and hippocampus more vulnerable to its effect. In case our in vitro data are confirmed in vivo in tissues from methylmalonic acidemic patients, it is feasible that that the present findings may be related to the pathophysiology of the tissue damage characteristic of these patients.