Ligand stabilization and effect on unfolding by polymorphism in human flavin-containing monooxygenase 3.

Pharmacogenomics is a powerful tool to prevent adverse reactions caused by different response of individuals to drug administration. Single nucleotide polymorphisms (SNPs) represent up to 90% of genetic variations among individuals. Drug metabolizing enzymes are highly polymorphic therefore the kinetic parameters of their catalytic reactions can be significantly influenced. This work reports on the unfolding process of a phase I drug metabolizing enzyme, human flavin-containing monooxygenase 3 (hFMO3) and its single nucleotide polymorphic variants (SNPs) V257M, E158K and E308G. Differential scanning calorimetry (DSC) indicates that the thermal denaturation of the enzyme is irreversible. The melting temperature (Tm) for the (Wild Type) WT and its polymorphic variants is found to be in a range from 46 °C to 50 °C. Also the activation energies of unfolding (Ea) show no significant differences among all proteins investigated (290-328 KJ/mol), except for the E308G variant that showed a significantly higher Ea of 412 KJ/mol. The presence of the bound NADP+ cofactor is found to stabilize all the variants by shifting the main Tm by 4-5 °C for all the proteins, exception made for E308G where no changes are observed. Isothermal titration calorimetry (ITC) was used to characterize the interaction of the protein with NADP+ in terms of dissociation constant (Kd), enthalpy (ΔH) and entropy (ΔS). Kd values of 1.6 and 0.7 µM, ΔH of -13.9 Kcal/mol and -16.8 Kcal/mol, ΔS of -20.5 cal/mol/deg, and -28.5 cal/mol/deg were found for V257M and E158K respectively. E308G was found to be unable to bind the NADP+ cofactor, a result that is in line with the Tm results. Circular dichroism also confirmed an overall lower stability of E308G, while NADP+ was found to give a strong positive shift of the Tm stabilizing the structure of E158K (46.2 to 50.6 °C). Previous data highlighted significant differences in terms of activity among the SNPs of hFMO3. In this work a minor impact of the SNPs was found on the stability of the enzyme in the ligand free form, except for E308G, whereas the binding of NADP+ reveals major differences among WT and polymorphic variants that are all measurable in terms of heat capacity, enthalpy and secondary structure content. These data provide the first direct evidence of ligand stabilization effects on hFMO3 that can explain the differences observed in catalytic efficiencies and serve as the starting point for the development of inhibitors of this enzyme.

["Banding" of the venous side of an arteriovenous fistula for hemodialysis complicated with steal syndrome: a clinical case]. / "Banding" del versante venoso di fistola artero-venosa per emodialisi complicata da sindrome di furto periferico: un caso clinico.

The duration of an arteriovenous fistula has a limit. In fact there are some complications that compromise a good working of them. We have dealed on of these complications, a steal syndrome of an omero-cephalic fistula by a simple operation of "banding" using a ring of Teflon around the arterialized vein getting a good clinical result with a good preservation of the blood flow trans-fistula.

Effects of uremia and dialysis on brain electrophysiology after recombinant erythropoietin treatment.

Quantitative electrophysiologic assessments are sensitive and useful indices of clinical state, and they are valuable in evaluating brain electrical activity before and after recombinant human erythropoietin (r-HuEPO) treatment. To study the hypothesis that, theoretically, anemia might be a cause of brain dysfunction in uremia, the authors assessed 18 patients (10 men and 8 women) on hemodialysis (RDT, age range, 35-58 years) before treatment (T1), and after 12 weeks (T2) and 24 weeks (T3) of r-HuEPO treatment, utilizing the following electrophysiologic tests: visual evoked potentials (VEP), brainstem auditory evoked responses (BAER), and somatosensory evoked potentials (SEP). The r-HuEPO was injected subcutaneously two times a week after RDT to produce hematocrit (Hct) levels of 30-35%. This drug induced a decrement of latency in P100 VEP (134.2 +/- 7.9 msec in T1 versus 116.5 +/- 6.9 msec in T2, p < 0.001, and versus 107.6 +/- 5.7 msec in T3, p < 0.005) and in the four main components of BAER. The most significant SEP changes were P27-N35 from peroneal nerve (p < 0.01), as an augmentation of SEP amplitude. Correction of anemia with r-HuEPO leads to a significant improvement in brain function in patients on RDT. The increased Hct level leads to enhanced brain oxygen delivery, directly improving brain metabolism. When the Hct rises, cerebral blood flow falls from high levels to normal, decreasing delivery of uremic "toxins" to the brain. The decrease in cerebral blood flow may decrease intracranial pressure and, in this way, may exert its beneficial effects by a rheologic pathway.

Clinical effects of recombinant human erythropoietin in hemodialysis patients. Results of the "Abruzzo" Multicenter Trial.

Anemia in regular dialysis treatment (RDT) patients is primarily due to a deficiency in renal-derived recombinant human erythropoietin (EPO). The aim of this study was to evaluate the results of a multicenter trial in 81 end-stage renal disease (ESRD) patients on RDT. An "open" study was conducted over 2 years; starting dose of r-HuEPO was 50 IU/kg/three times weekly i.v. and eventually was increased in steps of 25 Ul/kg/dialysis until 300 Ul/kg/week. Mean weekly dose per patient was 15 Ul/kg, with mean Hb increase of 27.5%. Mean hematocrit (Hct) levels increased in these patients from 22.9 +/- 2.5 to 31.7 +/- 2.8 (p less than 0.001) after 2 years of therapy. Both spontaneous and evoked potentials improved. The response to r-HuEPO is dose dependent; hypertension and hyperkalemia are the most common side effects, but they are easily controlled. Central nervous system function before and after treatment is improved, and seems consistent with an enhancement of patients' quality of life.

Therapeutic effects of simvastatin on hyperlipidemia in CAPD patients.

A causal link between hypercholesterolemia due to elevated plasma concentrations of LDL and VLDL remnants of CAPD patients has been established. The effects of 24 weeks of treatment with Simvastatin, a new HMG coenzyme A-reductase inhibitor (at 20 and 40 mg/day) on serum lipid, lipoprotein, and apolipoprotein A-I and B concentrations, as well as safety parameters and subjective side effects, were evaluated in eight patients (mean duration CAPD 24.80 +/- 7.50 months, age 54.50 +/- 13.70 years). Maximal effects on plasma lipoprotein and apolipoprotein concentrations were achieved after 4 weeks, and remained stable thereafter during the study. Mean fasting plasma cholesterol concentrations decreased from 280.5 +/- 60.2 mg% to 190.2 +/- 40.4 mg/dl (p less than 0.005) (-47%); mean plasma LDL-cholesterol concentrations also decreased from 257.6 +/- 13.4 mg% to 190.5 +/- 15.4 mg/dl (p less than 0.001) (-35%). Apolipoprotein A and B concentrations decreased significantly from 1.78 +/- 0.19 to 1.40 +/- 0.22 g/L (p less than .005) and 1.81 +/- 0.26 to 1.38 +/- 0.20 g/L (p less than .005). These data substantiate the view that Simvastatin is well tolerated and that no serious clinical or adverse laboratory effects have been observed. It appears to be a promising drug for the effective control of hyperlipemia in a large proportion of hypercholesterolemic patients, reducing their cardiovascular morbidity while on CAPD.