Marina Venzon Antunes

Oxidative stress and DNA damage in older adults that do exercises regularly

OBJECTIVES Free radicals may damage lipids, proteins and DNA, which may lead to critical diseases in the aging. This work evaluated levels of malondialdehyde (MDA), glutathione peroxidase (GPx) and DNA damage by comet assay (SCGE) in older adults that do exercises regularly. DESIGN AND METHODS 110 females, aged 66.3+/-8 years were divided into sedentary (n=54), walking (n=36) and muscle building (n=20) groups. Levels of MDA, GPx and SCGE were measured in venous blood before and after exercise. RESULTS MDA levels were higher (P<0.005) and GPx levels were lower (P<0.005) in active groups than in sedentary group. SCGE index after physical activity was greater than at baseline (muscle building: P=0.004; walking: P=0.002). CONCLUSIONS Exercise reduces the diseases risk, but may promote the production of free radicals. It remains unclear whether cell adaptations responsible for health benefits are associated with such events. However we may suggest the existence of a different biochemical pattern for older adults that do exercise regularly.

Simple procedure for determination of valproic acid in dried blood spots by gas chromatography-mass spectrometry.

Valproic acid (VA) is a drug widely used to treat epilepsy and bipolar disorder, at recommended serum concentrations ranging form 50 to 100μg ml(-1). A novel option for therapeutic drug monitoring that has been emerging recently its testing using dried blood spots on paper (DBS), but there are no reports of its application to assaying VA. In this study, a methodology was developed for the determination of VA in 6mm diameter DBS, equivalent to around 12μl of blood, using gas chromatography combined with mass spectrometry. DBS were extracted with a mixture of acetonitrile and methanol (1:3, v/v). The method is linear from 5 to 250μgml(-1) with intra-assay and inter-assay precision of 2.67-8.15% and 2.28-3.67%, respectively. Accuracy was 102.84-104.42%. VA was stable in DBS stored at 45°C for up to 21 days. VA concentrations in DBS correlated with concentrations assayed in serum, with r=0.9948. Mean ratio between VA concentrations in serum and DBS in clinical samples was 1.883. Dried blood spots are a viable option for collection and transport of samples and for assaying VA in the context of therapeutic drug monitoring, especially in Developing Countries.

Endoxifen levels and its association with CYP2D6 genotype and phenotype: evaluation of a southern Brazilian population under tamoxifen pharmacotherapy.

Background: An association between CYP2D6 variation and clinical outcomes among women with breast cancer treated with tamoxifen (TAM) has been demonstrated, such that the presence of 2 functional CYP2D6 alleles was associated with better clinical outcomes. This association is mainly due to the CYP2D6-mediated hydroxylation of N-desmethyltamoxifen (NDT) to yield endoxifen (EDF), which because of its high antiestrogenic potency, is mainly responsible for the therapeutic efficacy of TAM. The aim of this study was to evaluate the relation of CYP2D6 genotyping and phenotyping with EDF levels and [NDT]/[EDF] metabolic ratio in breast cancer patients from South of Brazil under TAM therapy. Methods: Trough blood samples were collected from 97 patients. CYP2D6 genotyping was performed with a luminex assay and calculation of genotypic activity scores. Tamoxifen and metabolites EDF, NDT, and 4-hydroxy-TAM were measured in plasma by high performance liquid chromatography with photo diode array detector. CYP2D6 phenotyping was performed by the determination of dextromethorphan (DMT) and dextrorphan (DTF) by high-performance liquid chromatography with fluorescence detection at plasma collected 3 hours after oral administration of 33 mg of DMF. Phenotypes were given according to [DMT]/[DTF] metabolic ratio. Results: CYP2D6 genotyping indicated a prevalence of 4.1% poor metabolizer, 4.1% intermediate metabolizer, 49.5% extensive metabolizer slow activity, 39.2% extensive metabolizer fast activity, and 3.1% ultrarapid metabolizer. Genotype (genotypic activity scores) was significantly correlated with phenotype ([DMT]/[DTF]), with a moderate association (rs = −0.463; P < 0.001). Median plasma concentrations (nanograms per milliliter; N = 97) were TAM 57.17; 4-hydroxy-TAM 1.01; EDF 6.21; NDT 125.50. EDF levels were lower in poor metabolizers than that in extensive metabolizers (P < 0.05). Phenotype showed stronger, but still moderate, association with EDF and [NDT]/[EDF] than genotype (r = −0.507, r = 0.625, P < 0.001 versus r = 0.356, r = 0.516, P < 0.01). Phenotype accounted for 26% of the variability in EDF levels and 38% of [NDT]/[EDF], whereas genotype accounted for 12% and 27%, respectively. Conclusions: CYP2D6 genotyping and/or phenotyping could not fully predict EDF concentrations. Monitoring EDF itself could be considered during TAM therapy.

Plasma concentrations of efavirenz are associated with body weight in HIV-positive individuals

BACKGROUND Efavirenz is among the most widely used antiretroviral drugs. Increased efavirenz exposure has been associated with CNS side effects and also with the chance of emergence of resistance upon treatment interruptions. The objective of this study was to evaluate factors associated with efavirenz plasma concentrations in a cohort of HIV-infected individuals. METHODS From July 2009 to March 2010, HIV-infected patients with efavirenz as part of antiretroviral therapy (600 mg at night), undetectable viral load for at least 1 year and CD4 cell count >200 cells/mm(3) were consecutively enrolled at the HIV/AIDS ambulatory care unit in southern Brazil. Plasma samples were taken 18-23 h after efavirenz last dose and analysed by validated ultra-performance liquid chromatography. RESULTS Forty-one subjects were included (21 females). Mean age and weight were 45.4 years and 70.9 kg, respectively. Mean efavirenz plasma concentration was 2.20 ± 2.17 mg/L. Most plasma concentrations (73%) were within the therapeutic window (1-4 mg/L); 17% were below and 10% above the limits. There were no significant associations between efavirenz concentration and age, CD4 cell count, time on antiretroviral treatment and gender. There was significant and inverse correlation between efavirenz concentrations and body weight (P = 0.013) and body mass index (P = 0.001). CONCLUSIONS In this cohort of well-controlled HIV-positive individuals, patients with lower weight or body mass index had a higher chance of presenting elevated plasma concentrations of efavirenz. Therapeutic drug monitoring to adjust dose might be a helpful tool to decrease efavirenz dose in order to minimize costs and adverse effects.

Clinical evaluation of a dried blood spot method for determination of mycophenolic acid in renal transplant patients.

OBJECTIVES The aim of this study is to evaluate the clinical application of dried blood spots (DBS) sampling in renal transplant patients under mycophenolic acid (MPA) immunosuppression, comparing measurements performed in paired plasma and DBS samples. DESIGN AND METHODS 77 paired DBS and plasma samples were obtained from 19 renal transplant patients. MPA was measured in both matrices by HPLC-DAD. Estimated plasma concentrations (EPC) were calculated from DBS concentrations (DC) using the formula EPC=DC/[1-(Hct/100)], using either individual or mean hematocrit (Hct). Agreement between methods was evaluated using Passing-Bablok regression and Bland-Altman difference plots. RESULTS MPA concentrations in DBS were in mean 60.7% of those measured in plasma. EPC calculated from DBS and patients individual Hct presented a high correlation with blood plasma (r=0.9862), and comparable absolute values (slope 1.0563 and intercept -0.0739), being in mean 102.2% of the measured plasma concentrations. EPC can also be calculated with the mean Hct of the group of patients, with similar results. CONCLUSIONS DBS sampling can be used for TDM of MPA in a clinical setting, employing conventional HPLC equipment, presenting similar results to plasma samples after a proper mathematical treatment. Moreover, due to its intrinsic stability and handling safety, DBS sampling can be considered a useful alternative especially in developing countries where sample logistics could be a major difficulty.

Estudo pré-analítico e de validação para determinação de malondialdeído em plasma humano por cromatografia líquida de alta eficiência, após derivatização com 2,4-dinitrofenilhidrazina

Malondialdehyde (MDA) is an important biomarker for the evaluation of oxidative stress status. The aim of this study was to develop a method for plasma MDA quantification by high performance liquid chromatography with diode-array detection (HPLC-DAD), following derivatization with 2,4-dinitrophenylhydrazine (DNPH), evaluating the main preanalytical variables. The calibration curve in plasma (0 to 40 µM) presented high linearity (r² = 0.998). Main validation parameters were: recovery: 78%; LOD: 0.11 µM and LOQ: 0.38 µM. The MDA values obtained in healthy volunteers (n=38) were 3.31 ± 0.38 µM (mean ± SD). Stability studies of MDA standard solution and derivatizing reagent and plasma MDA indicated that the standard solution can be stored at -20 and 4 oC, remaining stable for at least 30 days.

Development, validation and clinical evaluation of a dried urine spot method for determination of hippuric acid and creatinine.

OBJECTIVES The purpose of this study was to develop and validate a HPLC-DAD method for determination of creatinine and hippuric acid in dried urine spots (DUS), evaluating its clinical applicability. DESIGN AND METHODS Sample preparation was based on a simple one step extraction with water. Analysis was performed in a reversed phase column Hypersil Gold C18 (150 × 4.6 mm, 3 μm) with isocratic elution. Mobile phase was a mixture of triethylammonium phosphate buffer 5 mM, pH 3.3 and acetonitrile (90:10, v/v). RESULTS Total analytical run time was 6 min. Precision assays presented CV% lower than 11.8%. Accuracy was 96.0-103%. The lower limit of quantitation was 50 mg L(-1). Mean extraction yields were 99% for CR and 79.3% for HA. Analytes were stable in DUS up to 11 days at 40 °C. The method was tested in 49 paired DUS and urine samples, with acceptable agreement. HA-CR concentration ratios in DUS were in the range of 0.06 to 2.74, being in average 100.7% from those values found in urine. CONCLUSIONS DUS samples are a useful alternative for biological monitoring of toluene occupational exposure, especially in Developing Countries where sample logistics could be an important concern.

Monitoring imatinib plasma concentrations in chronic myeloid leukemia

Imatinib has proved to be effective in the treatment of chronic myeloid leukemia, but plasma levels above 1,000 ng/mL must be achieved to optimize activity. Therapeutic drug monitoring of imatinib is useful for patients that do not present clinical response. There are several analytical methods to measure imatinib in biosamples, which are mainly based on liquid chromatography with mass spectrometric or diode array spectrophotometric detection. The former is preferred due to its lower cost and wider availability. The present manuscript presents a review of the clinical and analytical aspects of the therapeutic drug monitoring of imatinib in the treatment of chronic myeloid leukemia. The review includes references published over the last 10 years. There is evidence that the monitoring of plasmatic levels of imatinib is an useful alternative, especially considering the wide pharmacokinetic variability of this drug.

CYP3A4*22 is related to increased plasma levels of 4-hydroxytamoxifen and partially compensates for reduced CYP2D6 activation of tamoxifen.

AIM To evaluate the impact of CYP3A4*22 in the formation of endoxifen (EDF) and hydroxytamoxifen (HTF), under different CYP2D6 genotypic backgrounds. MATERIALS & METHODS 178 patients were enrolled in the study. CYP2D6 and CYP3A4 genotyping and tamoxifen (TAM) and metabolites quantification were performed. RESULTS EDF concentrations were lower in poor (2.77 ng ml(-1)) and CYP2D6 intermediate metabolizers (5.84 ng ml(-1)), comparing to functional group (EM-F) (10.67 ng ml(-1), p < 0.001). HTF and TAM levels were respectively 47 and 53% higher in CYP3A4*22 carriers compared with *1/*1 patients in the whole group. Patients with impaired CYP2D6 metabolism and carriers of CYP3A4*22 had EDF levels comparable to CYP2D6 EM-F group (9.06 and 10.67 ng ml(-1), p = 0.247). CONCLUSION The presence of CYP3A4*22 might compensate the reduction of EDF concentrations related to CYP2D6 inactivity, especially due to increased HTF concentrations.

DBS sampling in imatinib therapeutic drug monitoring: from method development to clinical application

BACKGROUND Imatinib (IM) is widely used in treatment of chronic myeloid leukemia with target trough plasma concentrations above 1000 ng ml(-1). DBS can increase access to IM therapeutic drug monitoring. RESULTS IM was measured in the range 50-4000 ng ml(-1) by UHPLC-MS/MS using one 6 mm DBS in a fully validated method. IM was stable at DBS maintained at 40°C for 36 days. Plasma and DBS concentrations were highly correlated (r > 0.96). The use of a IM concentration target of 765 ng ml(-1) in DBS identified 93% of patients with plasma concentration below 1000 ng ml(-1). CONCLUSION IM can be measured in DBS using UHPLC-MS/MS with results comparable to those obtained in blood plasma.