Clara G. Dias

Quantification of the arylesterase activity of paraoxonase-1 in human blood

Paraoxonase-1 (PON1) is known as a free-radical scavenging system associated with circulating serum high-density lipoprotein (HDL). PON1 catalyzes the hydrolysis of multiple compounds such as arylesters, lactones and hydroperoxides. The arylesterase (AREase) activity of PON1 is involved in the detoxification of lipid peroxides, which are related to several clinical conditions. Therefore, the possibility of measuring the AREase activity in routine clinical studies would be advantageous. The AREase activity was obtained by monitoring the formation of acetic acid, upon the hydrolysis of phenyl acetate, using 10 μL of sample. The method accuracy was higher than 90% and intra-assay and inter-assay precisions were 96% and 95%, respectively. The method validation supported that this analytical procedure is suitable for use in human serum and heparinized plasma samples, while ethylenediaminetetra-acetic acid (EDTA)-containing samples should be avoided. The methodology herein described constitutes an easy, fast and reliable method for assessing the AREase activity of PON1. This method can be easily implemented as a clinical analytical tool and is also suitable for research purposes.

Development and validation of an HPLC-UV method for quantifying nevirapine and its main phase I metabolites in human blood

Nevirapine (NVP) is a widely used drug for the treatment of HIV-infection, which is associated with severe toxic events dependent on its biotransformation. Thus, the availability of analytical methods for the biomonitoring of NVP and its main metabolites is of utmost importance. In this paper we report the development and validation of a reversed phase HPLC-UV method for the quantification of NVP and its main phase I metabolites in human plasma. The method was validated over a range of 10–2500 ng mL−1 for the phase I metabolites of NVP and 10–10 000 ng mL−1 for NVP. The coefficients of variation (CV) of the average back-calculated concentrations were lower than 9% and the LLOQ was 10 ng mL−1 for each analyte. The accuracy and precision of the method were acceptable. There was no significant interference from plasma components or from other typically co-administered antiretroviral drugs and the mean recovery was 94%. This method represents an inexpensive, sensitive, accurate and precise alternative to mass spectrometry-based biomonitoring of NVP.

Nevirapine modulation of paraoxonase-1 in the liver: An in vitro three-model approach.

INTRODUCTION Nevirapine is associated with severe hepatotoxicity, through the formation of reactive metabolites. Paraoxonase-1 (PON-1) is a promiscuous enzyme involved in the metabolism of xeno- and endobiotics and proposed as a biomarker of hepatotoxicity. The aim of this work was to explore the effects of nevirapine and its phase I metabolites, 2-hydroxy-nevirapine and 12-hydroxy-nevirapine, on PON-1 activities. MATERIAL AND METHODS 2D and 3D primary cultures of rat hepatocytes, and also HepG2 2D cell cultures, were exposed to nevirapine, 2-hydroxy-nevirapine, and 12-hydroxy-nevirapine. The paraoxonase (POase), arylesterase (AREase) and lactonase (LACase) activities of PON-1 were quantified. RESULTS Effects of nevirapine and its metabolites were only observed in the 3D cell model. Both nevirapine and 12-hydroxy-nevirapine increased POase (p<0.05, p<0.01) and LACase activities (p<0.05, p<0.001). The AREase activity was increased only upon 12-hydroxy-nevirapine exposure (p<0.01). These modulatory effects were observed at 300μM concentrations of nevirapine and 12-hydroxy-nevirapine. CONCLUSIONS The formation of 12-hydroxy-nevirapine seems to be the main factor responsible for the increase of PON-1 activities induced by nevirapine exposure. This effect was only observed in the 3D model, suggesting that an in vivo-like system is necessary for this modulation to occur. The present data suggest that the 3D model is a more suitable in vitro model than the conventional ones to explore drug effects on PON-1.

Monitoring of the lactonase activity of paraoxonase-1 enzyme in HIV-1-infection

Paraoxonase‐1 (PON1) is a high‐density lipoprotein (HDL)‐associated enzyme known as a free radical scavenging system ( 1 ). PON‐1 has three main activities, responsible for its antioxidant and anti‐inflammatory potential: paraoxonase, arylesterase and lactonase (LACase), the latest to be discovered and pointed out to be its native activity ( 2 ). Among other physiological roles, the LACase might minimize the deleterious effects of hyperhomocysteinaemia in infection, by detoxifying the highly reactive metabolite homocysteine‐thiolactone (HcyTL) ( 3 ), 4 . In the present work, we have developed and applied a method to quantify LACase activity and to explore the role of this enzyme in HIV‐infection and virological response. The LACase activity was monitored in a cohort of HIV‐1‐infected patients, through the titration of 3‐(o‐hydroxyphenyl) propionic acid, formed upon the LACase‐mediated hydrolysis of the substrate dihydrocoumarin. The study protocol was approved by the Ethics Committee of Centro Hospitalar de Lisboa Central and Hospital Prof. Doutor Fernando Fonseca. All patients gave their written informed consent and were adults with documented HIV‐1‐infection, regardless of combined antiretroviral therapy (cART) use. Naïve patients and patients who had received continuous antiretroviral treatment for more than one month were included. A total of 179 HIV‐1‐infected patients were included on this study (51% Men, 39% non‐Caucasian, 45±13 years old). Patients with non‐suppressed viraemia, either from the non‐cART (n=89, 12±4 kU/L, p<0.01) or from the cART with detectable viral load (n=11, 10±5 kU/L, p<0.05) groups, had lower activity than the cART with suppressed viraemia (n=79, 15±7 kU/L) (Kruskal–Wallis test). Among naïve patients, higher viral load (> 31,500 cps/mL, Spearman r=−0.535, p=0.003) and lower CD4+ T‐cells count (< 500 cell/mm3, Pearson r=0.326, p=0.024) were associated with the LACase activity. The present study suggests that lower LACase activity is associated with uncontrolled HIV‐1‐infection, particularly with non‐suppressed viraemia, despite of cART. This data seems to point to LACase role in HIV‐infection, probably reflecting an increased formation of HcyTL deleterious species. A better knowledge of the LACase and its role in HcyTL pathophysiology might identify new therapeutic targets in HIV‐1‐infected patients.

Cysteine Oxidative Dynamics Underlies Hypertension and Kidney Dysfunction Induced by Chronic Intermittent Hypoxia

Previous data showed the lack of efficacy of an adrenoceptor antagonist to revert hypertension induced by chronic intermittent hypoxia (CIH). We hypothesized that, in addition to sympathetic activation, CIH may change the availability and dynamics of cysteine. Temporal variation in total cysteine and its fractions, free reduced, free oxidized and protein-bound (CysSSP), were measured in homogenates of kidney cortex and medulla of Wistar rats. Animals were exposed to CIH for 14, 21 and 60 days and cysteine fractions and fibronectin gene expression were assessed at these time-points. Two different phases in cysteine dynamics were identified. An early phase (14d) characterized by an increase in cysteine oxidation and CysSSP forms. Late events (>21d) were characterized by a global reduction in cysteine, minimum level of CysSSP and maximum overexpression of fibronectin in kidney cortex. In conclusion, cysteine dynamics is influenced by the duration of CIH exposure: first there is a cysteine disulfide stress-like adaptive response followed by a progressive loss of cysteine availability and a decrease in CysSSP fraction. Kidney fibrosis associated to an unbalance in cysteine dynamics might contribute to the inefficacy of available antihypertensive drugs in patients with delayed diagnosis of sleep apnea.

Assessment of human paraoxonase activity by electrochemistry: a simple and novel approach

Human serum paraoxonase 1 (EC 3.1.8.1, PON1), a calcium dependent enzyme, is an endogenous free-radical scavenging system with arylesterase, lactonase and paraoxonase activities. The determination of PON1 activity has been gaining an increasing role in clinical diagnosis due to its possible relationship with atherosclerosis and derived diseases. The paraoxonase activity protects against xenobiotic toxicity and was the first to be discovered. It has been the most used activity to assess PON1 status, through the spectrophotometric measurement of the hydrolysis of organophosphate compounds, such as paraoxon. However, these methods are prone to interferences and require specialized equipment. Herein, a simple alternative electrochemical assay for the assessment of PON1 activity is proposed for the first time. The catalytic hydrolysis of paraoxon was monitored by square-wave voltammetry, and the kinetic parameters of Escherichia coli expressed PON1 variant G3C9 (Vappmax 530 ± 40 nM s−1, KappM 1.7 ± 0.4 mM) and native PON1 (Vappmax 390 ± 40 nM s−1, KappM 1.3 ± 0.3 mM), present in human plasma, were determined.

Sex differences in apolipoprotein A1 and nevirapine-induced toxicity.

Nevirapine (NVP) is associated with severe liver and skin toxicity through sulfotransferase (SULT) bioactivation of the phase I metabolite 12‐hydroxy‐NVP [ 1 – 3 ]. The female sex, a well‐known risk factor for NVP‐induced toxicity, is associated with higher SULT expression [ 4 ] and lower plasma levels of 12‐hydroxy‐NVP [ 3 ]. Interestingly, apolipoprotein A1 (ApoA1) increases SULT2B1 activity and ApoA1 synthesis is increased by NVP [ 5 , 6 ]. Herein, we explore the effect of ApoA1 levels on NVP metabolism and liver function. The study protocol was firstly approved by the hospitals’ Ethics Committees. All included individuals were HIV‐infected patients treated with NVP for at least one month. The plasma concentrations of NVP and its phase I metabolites were quantified by HPLC [ 7 ]. ApoA1 levels were assessed by an immunoturbidimetric assay. Forty‐nine HIV‐infected patients on NVP were included (53% men, 59% Caucasian). NVP plasma levels were correlated with HDL‐cholesterol (Spearman r=0.2631; p=0.0441) and ApoA1 (Spearman r=0.3907; p=0.0115). Women had higher ApoA1 levels than men (Students t Test; p=0.0051). In both sexes, 12‐hydroxy‐NVP levels were negatively correlated with ApoA1 (male: Spearman r=−0.3810; p=0.0499 female: Spearman r=−0.5944; p=0.0415). In men, ApoA1 was positively correlated with aspartate aminotransferase (AST, Spearman r=0.5507; p=0.0413), while in women ApoA1 was associated (Spearman r=0.6408; p=0.0056) with alanine aminotransferase (ALT). These results show sex differences in NVP‐induced ApoA1 synthesis. The higher ApoA1 levels in women might stabilize SULT2B1 [ 6 ]. This would explain the lower levels of 12‐hydroxy‐NVP [ 3 ] and the higher hepatotoxicity found in women, due to increased sulfonation of this metabolite. These data support a role for ApoA1 in the sex dimorphic mechanism leading to NVP‐induced toxicity.