4 (range 15–30 mg/kg/day, given orally once a day) The mean dura

4 (range 15–30 mg/kg/day, given orally once a day). The mean duration of HU was 2.12 ± 1.49 years. Dose escalation was guided by clinical and hematological response with no attempt to reach the maximum tolerated dose (MTD);16 3 subjects were at MTD at time of enrollment. RGFP966 Blood samples for determination of MDA, nitrite, PON, TAO, and vitamin E were collected and processed as follows: 5 mL of blood were collected into plain tubes and allowed to clot for 30 min at 25 °C; it was then centrifuged at 3,000 rpm for

15 min at 4 °C, and the serum was separated into clean, properly labeled tubes for analysis. Determination of lipid peroxidation: Lipid peroxidation was assayed by measuring the level of MDA. It was determined by measuring thiobarbituric reactive species using the method of Ruiz-Larrea et al., 17 in which the thiobarbituric acid-reactive substances react with thiobarbituric acid to produce a red colored complex with peak absorbance OTX015 molecular weight at 532 nm. Determination of serum nitrite: Serum nitrite (NO2−), as an acceptable surrogate marker to serum NO, was measured using Griess reagent, following the Moshage et al. method; 18 nitrite, a stable end-product of NO radical, is commonly used as indicator for the production of NO. 19 Determination of PON activity:

Arylesterase activity of PON was measured spectrophotometrically in supernatants using phenylacetate as a substrate. 20 Measurement of serum TAO levels: Serum TAO levels were determined using an automated measurement method, which is based on the bleaching of the characteristic color of a more stable 2, 2-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid, [ABTS]) radical cation by antioxidants (Beckman Coulter – Fullerton, CA, USA). 21 The ABTS radical cation is decolorized by antioxidants according to their concentrations and antioxidant capacities. The results are expressed in mmol Trolox equivalents/L. Measurement of vitamin E: Vitamin E as tocopherol was measured by HPLC. 22 Freshly-obtained erythrocytes were stored in Niclosamide 2% pyrogallol in ethanol

at -70 °C. All samples were analyzed within one month of storage using a reverse-phase C-18 column (Waters – Milford, MA, USA), a 95% methanol solvent system, and a UV/VIS detector set at 292 nm. 22 Patients’ data were analyzed using the Statistical Package for Social Sciences (SPSS), version 17.0 for Windows. Quantitative variables were expressed by mean ± standard deviation (SD), and compared using Student’s t-test for unpaired samples and Mann–Whitney’s test. Spearman’s rank-order test was used for correlating quantitative variables. Qualitative variables were expressed as numbers (frequency) and percentages, and compared between groups using the chi-squared test. Logistic regression analysis was performed, and accuracy, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated. A receiver operating characteristic (ROC) curve was made, and area the under the curve (AUC) was calculated.

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