Stopped-flow Light Scattering Analysis of Red Blood Cell Glycerol Permeability.

Stopped-Flow Light Scattering (SFLS) is a method devised to analyze the kinetics of fast chemical reactions that result in a significant change of the average molecular weight and/or in the shape of the reaction substrates. Several modifications of the original stopped-flow system have been made leading to a significant extension of its technical applications. One of these modifications allows the biophysical characterization of the water and solute permeability of biological and artificial membranes. Here, we describe a protocol of SFLS to measure the glycerol permeability of isolated human red blood cells (RBCs) and evaluate the pharmacokinetics properties (selectivity and potency) of isoform-specific inhibitors of AQP3, AQP7 and AQP9, three mammalian aquaglyceroporins allowing transport of glycerol across membranes. Suspensions of RBCs (1% hematocrit) are exposed to an inwardly directed gradient of 100 mM glycerol in a SFLS apparatus at 20 °C and the resulting changes in scattered light intensity are recorded at a monochromatic wavelength of 530 nm for 120 s. The SFLS apparatus is set up to have a dead time of 1.6-ms and 99% mixing efficiency in less than 1 ms. Data are fitted to a single exponential function and the related time constant (τ, seconds) of the cell-swelling phase of light scattering corresponding to the osmotic movement of water that accompanies the entry of glycerol into erythrocytes is measured. The coefficient of glycerol permeability ( Pgly , cm/s) of RBCs is calculated with the following equation: Pgly = 1/[(S/V)τ] where τ (s) is the fitted exponential time constant and S/V is the surface-to-volume ratio (cm-1) of the analyzed RBC specimen. Pharmacokinetics of the isoform-specific inhibitors of AQP3, AQP7 and AQP9 are assessed by evaluating the extent of RBC Pgly values resulting after the exposure to serial concentrations of the blockers.

Role of folic acid depletion on homocysteine serum level in children and adolescents with epilepsy and different MTHFR C677T genotypes.

Homocysteine (Hcy) is a sulfur-containing amino acid involved in methionine metabolism. An elevated total plasma Hcy concentration (tHcy) is a risk factor for vascular disease. The present study aimed to assess the role of antiepileptic drugs (AEDs) and C677T methylenetetrahydrofolate (MTHFR) polymorphisms on tHcy in pediatric patients with epilepsy treated for at least 6 months with various treatment regimens protocols including the newer AEDs. The study group was recruited from children and adolescents with epilepsy followed up in the Child Neuropsychiatry Clinic of the Second University of Naples, between January 2007 and March 2008. Inclusion criteria were: (1) patients with epilepsy, treated with one or more anticonvulsant drugs for at least 6 months; (2) age between 2 and 16 years. Plasma tHcy concentrations were considered elevated when they exceeded 10.4 µmol/L, and folate concentrations <3 ng/mL were considered deficient. Serum vitamin B12 levels were considered normal between 230 and 1,200 pg/mL. The study group was composed of 78 patients (35 males, 43 females), aged between 3 and 15 years (mean 8.9 years). Thirty-five patients were taking AED monotherapy, 43 polytherapy. Sixty-three healthy sex- and age-matched children and adolescents served as controls. The mean tHcy value in the patient group was higher than the mean value in the control group (12.11 ± 7.68 µmol/L vs 7.4±4.01 µmol/L; p<0.01). DNA analysis for the MTHFR C677T polymorphism showed the CT genotype in 46%, CC in 35% and TT in 17.8% of cases. Decreased folic acid serum levels significantly correlated with increased tHcy levels (p<0.003). Female sex was a less significant risk factor for increased tHcy levels (p=0.039). Our study confirms the association between hyperhomocysteinemia and epilepsy. The elevation of tHcy is essentially related to low folate levels. Correction of poor folate status, through supplementation, remains the most effective approach to normalize tHcy levels in patients on AED mono- or polytherapy.

Insulin sensitivity in prepubertal Caucasian normal weight children.

BACKGROUND: Determining whether or not a child is insulin resistant is becoming a matter of fundamental interest even in childhood, as the incidence of metabolic complications of insulin resistance is rising. Thus reference values are needed. AIM: To set percentiles for HOMA-IR and WBISI in Caucasian normal weight prepubertal children. POPULATION: 238 normal weight prepubertal Caucasian children (126 males/112 females). METHODS: After physical examination including anthropometric measurements all children underwent an oral glucose tolerance test from which HOMA-IR and WBISI were calculated. RESULTS: HOMA-IR values above the 95th percentile (2.03) and WBISI values below the 5th percentile (5.67) represent the cut-off values for the determination of insulin resistance. CONCLUSION: These reference values are useful to easily detect insulin resistance within the prepubertal Caucasian population representing a helpful tool for clinicians to target such children in order to improve insulin sensitivity.

Correlations between phenotype and microsatellite instability in HNPCC: implications for genetic testing.

Hereditary nonpolyposis colorectal cancer (HNPCC) is widely considered to be a syndrome of defective mismatch repair (MMR). A major concern with genetic diagnosis of HNPCC is the variable, often low, percentage of pathogenic germline mutations that can be detected in MMR genes using common screening methods. The variable percentage of mutation detected is in part related to the sensitivity of conventional screening methods and may also depend on the heterogeneous genetics of HNPCC. Thus, identification of phenotypic criteria predictive of germline mutations in MMR genes may be helpful in efficient HNPCC genetic testing. Clinical diagnostic criteria, initially developed for HNPCC (e.g., Amsterdam I and II, or Bethesda criteria), can be used to clinically select patient candidates that carry germline mutations in MMR genes. More useful criteria were previously developed by analyzing families with germline MMR mutations. Using a complementary approach based on tumor microsatellite instability analysis, we confirm that the Amsterdam criteria are significantly better than the Bethesda criteria in predicting families with MSI-H tumors (P = 0.0227). Our results also suggest that a cutoff at < 50 years' mean age at diagnosis of HNPCC-related cancers (especially colorectal and endometrial cancer) may be an additional tool for the identification of families with defective MMR. Recent advances in MMR mutation screening are expected to improve detection of pathogenic MMR mutations in these families. Conversely, the high proportion of MSS tumors observed in our series of families with advanced age at cancer diagnosis is consistent with the low percentage of MMR mutations detected by previous studies in families with this phenotype. These families probably carry mutations in other genes that may or may not be related to MMR. Additional studies are necessary to clarify the molecular basis for HNPCC in families with MSS tumors.