Tumescent local anesthesia with ropivacaine in different concentrations in bitches undergoing mastectomy: plasma concentration and post-operative analgesia

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Influence of training status and eNOS haplotypes on plasma nitrite concentrations in normotensive older adults: a hypothesis-generating study

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Are plasma levels valid surrogates for cellular concentrations of antiretroviral drugs in HIV-infected patients?

Total plasma concentrations are currently measured for therapeutic drug monitoring of HIV protease inhibitors (PIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs). However, the pharmacological target of antiretroviral drugs reside inside cells. To study the variability of their cellular accumulation, and to determine to which extent total plasma concentrations (TPC) correlate with cellular concentrations (CC), plasma and peripheral blood mononuclear cells (PBMCs) were simultaneously collected at single random times after drug intake from 133 HIV infected patients. TPC levels were analysed by high-performance liquid chromatography with ultraviolet detection and CC by LC-MS/MS from peripheral blood mononuclear cells. The best correlations between TPC and CC were observed for nelfinavir (NFV, slope=0.93, r=0.85), saquinavir (SQV, slope=0.76, r=0.80) and lopinavir (LPV, slope=0.87, r=0.63). By contrast, TPC of efavirenz (EFV) exhibited a moderate correlation with CC (slope=0.69, r=0.58), while no correlation was found for nevirapine (NVP, slope=-0.3, r=0.1). Interindividual variability in the CC/TPC ratio was lower for protease inhibitors (coefficients of variation 76%, 61%, and 80% for SQV, NFV and LPV, respectively) than for nonnucleoside reverse transcriptase inhibitors (coefficients of variation 101% and 318%, for EFV and NVP). As routine CC measurement raises practical difficulties, well-correlated plasma concentrations (ie, NFV, SQV and LPV) can probably be considered as appropriate surrogates for cellular drug exposure. For drugs such as EFV or NVP, there may be room for therapeutic drug monitoring improvement using either direct CC determination or other predictive factors such as genotyping of transporters or metabolizing enzyme genes.

(Table 1) Hepatic EROD activity and and retinoid concentrations in the liver, and plasma thyroid hormones of northern fulmars (Fulmarus glacialis) from Bjørnøya

We investigated whether the hepatic cytochrome P450 1A activity (measured as 7-ethoxyresorufin-O-deethylase (EROD)) and plasma thyroid hormone and liver retinoid concentrations were explained by liver and blood levels of halogenated organic contaminants (HOCs) in free-ranging breeding northern fulmars (Fulmarus glacialis) from Bjornoya in the Norwegian Arctic. Hepatic EROD activity and liver levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalents (TEQs) were positively correlated, suggesting that hepatic EROD activity is a good indicator for dioxin and dioxin-like HOC exposure in breeding northern fulmars. There were not found other strong relationships between HOC concentrations and hepatic EROD activity, plasma thyroid or liver retinoid concentrations in the breeding northern fulmars. It is suggested that the HOC levels found in the breeding northern fulmars sampled on Bjornoya were too low to affect plasma concentrations of thyroid hormones and liver levels of retinol and retinyl palmitate, and that hepatic EROD activity is a poor indicator of polychlorinated biphenyl (PCB) and pesticide exposure.

Comparative Study of the Changes in Partial Pressure of Plasma Carbon Dioxide During Carbon Dioxide Insufflation into the Intraperitoneal and Preperitoneal Spaces

Background: We aimed to compare plasma concentrations of carbon dioxide (CO(2)) in dogs that underwent intra- and preperitoneal CO(2) insufflation. Materials and Methods: Thirty dogs were studied. Ten formed a control group, 10 underwent intraperitoneal CO(2) insufflation, and 10 underwent preperitoneal CO(2) insufflation. General anesthesia with controlled ventilation was standardized for all dogs. After stabilizing the anesthesia, blood samples were collected at predetermined times and were sent for immediate gasometric analysis. Analysis of variance was used for comparing variables. Results: The plasma CO(2) concentration in the intraperitoneal insufflation group increased significantly more than in the preperitoneal insufflation group and was significantly greater than in the control group (P < 0.05). The pH values in the intraperitoneal group were lower than in the preperitoneal group (P < 0.05). Conclusion: The data from this study suggest that a greater plasma concentration of CO(2) is achieved by insufflation at constant pressure into the intraperitoneal space than into the preperitoneal space.

Improved high-performance liquid chromatography determination of methotrexate and its major metabolite in plasma using a poly(styrene-divinylbenzene) column

A sensitive high-performance liquid chromatographic assay has been developed for measuring plasma concentrations of methotrexate and its major metabolite, 7-hydroxymethotrexate. Methotrexate and metabolite were extracted from plasma using solid-phase extraction. An internal standard, aminopterin was used. Chromatographic separation was achieved using a 15-cm poly(styrene-divinylbenzene) (PRP-1(R)) column. This column is more robust than a silica-based stationary phase. Post column, the eluent was irradiated with UV light, producing fluorescent photolytic degradation products of methotrexate and the metabolite. The excitation and emission wavelengths of fluorescence detection were at 350 and 435 nm, respectively. The mobile phase consisted of 0.1 M phosphate buffer (pH 6.5), with 6% N,N-dimethylformamide and 0.2% of 30% hydrogen peroxide. The absolute recoveries for methotrexate and 7-hydroxymethotrexate were greater than 86%. Precision, expressed as a coefficient of variation (n=6), was

Homocysteine and cysteine concentrations are modified by recent exposure to environmental air pollution in Sao Paulo, Brazil

Millions of people worldwide are affected by anthropogenic air pollution derived from the combustion of fossil fuels. In this work, we tested the effects of fetal, lactation and post-weaning ambient air pollution exposure on total homocysteine (tHcy) concentrations and on a downstream pathway element, the plasma cysteine (Cys) concentration. Two similar exposure chambers (polluted and filtered chamber) were located near an area with heavy traffic in Sao Paulo, Brazil, and male Swiss mice were housed there from the pre-natal period until 3 months of age. Groups during fetal, lactation and adult periods of exposure were apportioned, and tHcy and Cys plasma concentrations were assessed when the animals were 3 months old. In our study, both the tHcy and Cys concentrations were decreased in groups that spent their final stage of life in polluted chambers, suggesting recent alterations in tHcy and Cys concentrations due to air pollution exposure. The possible relationship of these data with cardiovascular dysfunction is still a matter of controversy in animals; nevertheless, epigenetic mechanisms emerge as a possible issue to consider in the investigation of the link between air pollution and Hcy measurement. (C) 2009 Elsevier Inc. All rights reserved.

Enhanced concentrations of relevant markers of nitric oxide formation after exercise training in patients with metabolic syndrome

Metabolic syndrome (MetS) denotes a clustering of risk factors that may affect nitric oxide (NO) bioavailability and predispose to cardiovascular diseases, which are delayed by exercise training. However, no previous study has examined how MetS affects markers of NO formation, and whether exercise training increases NO formation in MetS patients. Here, we tested these two hypotheses. We studied 48 sedentary individuals: 20 healthy controls and 28 MetS patients. Eighteen MetS patients were subjected to a 3-month exercise training (E+group), while the remaining 10 MetS patients remained sedentary (E-group). The plasma concentrations of nitrite, cGMP, and ADMA (asymmetrical dimethylarginine: an endogenous nitric oxide synthase inhibitor), and the whole blood nitrite concentrations were determined at baseline and after exercise training using an ozone-based chemiluminescence assay, and commercial enzyme immunoassays. Thiobarbituric acid reactive species (TBA-RS) were measured in the plasma to assess oxidative stress using a fluorometric method. We found that, compared with healthy subjects, patients with MetS have lower concentrations of markers of NO formation, including whole blood nitrite, plasma nitrite, and plasma cGMP, and increased oxidative stress (all P < 0.05). Exercise training increased the concentrations of whole blood nitrite and cGMP, and decreased both oxidative stress and the circulating concentrations of ADMA (both P < 0.05). These findings show clinical evidence for lower endogenous NO formation in patients with MetS, and for improvements in NO formation associated with exercise training in MetS patients. (C) 2008 Elsevier Inc. All rights reserved.

Interethnic differences in ADMA concentrations and negative association with nitric oxide formation in preeclampsia

Background: Recent studies have suggested that impaired nitric oxide (NO) formation in preeclampsia may result from increased concentrations of an endogenous NO synthase inhibitor, the asymmetric dimethylarginine (ADMA). However, no previous study has examined whether a negative association exists between ADMA and nitrite concentrations in preeclampsia. Moreover, no previous study has compared ADMA and nitrite levels in black and white preeclamptic pregnant women. Methods: We measured plasma nitrite concentrations using an ozone-based chemiluminescence assay, and plasma ADMA levels using enzyme immunoassays in 94 pregnant (47 healthy pregnant: 16 blacks and 31 whites; and 47 preeclamptic: 14 blacks and 33 whites). Results: We found higher ADMA (2.199 +/- 0.016 mu mol/l vs. 2.112 +/- 0.012 mu mol/l; P < 0.0001) and lower plasma nitrite levels (102 +/- 7.1 nmol/l vs. 214.8 +/- 26.1 nmol/l; P<0.0001) in preeclamptic compared with healthy pregnant women. Black pregnant had higher ADMA levels than white pregnant women (P<0.05), both in preeclamptic (2.239 +/- 0.020 mu mol/l vs. 2.144 +/- 0.019 mu mol/l) and in healthy pregnant (2.172 +/- 0.025 mu mol/l vs. 2.077 +/- 0.018 mu mol/l). Conversely, we found no significant effects of ethnicity on the plasma nitrite levels, both in healthy pregnant and in preeclamptic women (P>0.05). We found a significant negative correlation (P<0.05) between these markers (r = 0.28; P<0.05). Conclusions: Our findings show higher ADMA and lower nitrite levels in preeclamptic compared with healthy pregnant, and the concentrations of these biomarkers are inversely associated. While ethnicity affected ADMA concentrations, no such effect was found with respect to nitrite levels. These results may have important implications for studies on NO biology and therapeutic approaches of preeclampsia. (C) 2010 Elsevier B.V. All rights reserved.

Elevated progesterone concentrations enhance prostaglandin F(2 alpha) synthesis in dairy cows

The objective was to evaluate the influence of varying plasma progesterone (P(4)) concentrations throughout the luteal phase in dairy cows on PGF(2 alpha) production (assessed as plasma concentrations of 13,14-dihydro-15-keto-PGF(2 alpha); PGFM) following treatment with estradiol-17 beta (E(2)) or oxytocin (OT). In all experiments, time of ovulations was synchronized with the OvSynch protocol and Day 0 corresponded to day of second GnRH injection. In Experiment 1, non-lactating dairy cows on Day 6 remained non-treated (n = 9), received 20 mg LH (n = 7), or had ovarian follicles larger than 6 mm aspirated (n = 8). In Experiment 2, cows on Day 6 were untreated (n = 9) or received 5000 IU hCG (n = 10). In Experiments 1 and 2, all cows received 3 mg E(2) on Day 17, and blood samples were collected every 30 min from 2h before to 10h after E(2). Experiment 3 was conducted in two periods, each from Days 0 to 17 of the estrous cycle. At the end of Period 1, animals switched treatments in a crossover arrangement. Animals in Group 2/8 (n = 4) received 2 kg/d of concentrate in the first period and 8 kg/d in the second period. Animals in Group 8/2 (n = 7) received the alternate sequence. Blood was collected daily for measurement Of P(4) 4 h after concentrate feeding. On Day 17, blood was collected from 1 h before to 1 h after a 100 IU OT injection. In Experiment 1, both plasma P(4) and release Of PGF(2 alpha) were similar between LH-treated and control cows (P > 0.10). In Experiment 2, plasma P4 was elevated to a greater extent on Day 17 in cows treated with hCG (P < 0.05) and plasma PGFM was also greater in hCG-treated animals (treatment x time interaction; P < 0.05). In Experiment 3, there was a group x period interaction (P < 0.01) for plasma P(4), indicating that less concentrate feeding was associated with greater plasma P(4). Release of PGF(2 alpha) in response to OT was greater for cows receiving less concentrate (group x period interaction; P < 0.05). In conclusion, dairy cows with more elevated blood P(4) concentrations released more PGF(2 alpha) in response to E(2) or OT. (c) 2008 Elsevier B.V. All rights reserved.

Inverse relationship between markers of nitric oxide formation and plasma matrix metalloproteinase-9 levels in healthy volunteers

Background: Nitric oxide (NO) is a major regulator of cardiovascular homeostasis and has anti-atherogenic properties. Reduced NO formation is associated with endothelial dysfunction and with cardiovascular risk factors. Although NO downregulates the expression and activity of the pro-atherogenic enzyme matrix metalloproteinase-9 (MMP-9), no previous clinical study has examined whether endogenous NO formation is inversely associated with the circulating levels of pro-MMP-9, which are associated with cardiovascular events. We examined this hypothesis in 175 healthy male subjects who were non-smokers. Methods: To assess NO bioavailability, the plasma concentrations of nitrite, nitrate, and cGMP were determined using an ozone-based chemiluminescence assay and an enzyme immunoassay. Pro-MMP-9 and pro-MMP-2 levels were measured in plasma samples by gelatin zymography. Results: We found significant negative correlations between pro-MMP-9 levels and plasma nitrite (P=0.035, rs=-0.159), nitrate (P=0.040, rs=-0.158), and cGMP (P=0.011, rs=-0.189) concentrations. However, no significant correlations were found between pro-MMP-2 levels and the plasma concentrations of markers of NO bioavailability (all P>0.05). Conclusions: There is an inverse relationship between markers of NO formation and plasma MMP-9 levels. This finding may shed some light on the possible mechanisms involved in the increased cardiovascular risk of apparently healthy subjects with low NO bioavailability or high circulating levels of pro-MMP-9. (C) 2008 Elsevier B.V. All rights reserved.

Plasma growth hormone and growth hormone-binding protein during development in the marsupial brushtail possum (Trichosurus vulpecula)

Plasma concentrations of growth hormone (GH) were measured in the brushtail possum (Trichosurus vulpecula) pouch young from 25 through to 198 days post-partum (n=71). GH concentrations were highest early in pouch life (around 100 ng/ml), and thereafter declined in an exponential fashion to reach adult concentrations (10.8 +/- 1.8 ng/ml; n=21) by approximately 121-145 days post-partum, one to two months before the young is weaned. Growth hormone-binding protein (GHBP), which has been shown to modify the cellular actions of GH in eutherian mammals, was identified for the first time in a marsupial. Based on size exclusion gel filtration, possum GHBP had an estimated molecular mass of approximate to 65 kDa, similar to that identified in other mammalian species, and binding of I-125-labelled human GH (hGH) was displaced by excess hGH (20 mug). An immunoprecipitation method, in which plasma GHBP was rendered polyethylene glycol precipitable with a monoclonal antibody to the rabbit GHBP/GH receptor (MAb 43) and labelled with I-125-hGH, was used to quantitate plasma GHBP by Scatchard analysis in the developing (pooled plasma samples) and adult (individual animals) possums. Binding affinity (K-a) values in pouch young aged between 45 and 54 and 144 and 153 days post-partum varied between 1.0 and 2.4 x 10(9)/M, which was slightly higher than that in adult plasma (0.96 +/- 0.2 x 10(9)/M, n = 6). Binding capacity (B-max) values increased from non-detectable levels in animals aged 25-38 days post-partum to reach concentrations around half that seen in the adult (1.4 +/- 0.2 x 10(-9) M) by about 117 days post-partum and remained at this level until 153 days post-partum. Therefore, in early pouch life when plasma GH concentrations are highest, the very low concentrations of GHBP are unlikely to be important in terms of competing with GH-receptor for ligand or altering the half-life of circulating GH.

Multiplex ultra-performance liquid chromatography-tandem mass spectrometry method for simultaneous quantification in human plasma of fluconazole, itraconazole, hydroxyitraconazole, posaconazole, voriconazole, voriconazole-N-oxide, anidulafungin, and caspofungin.

Therapeutic drug monitoring (TDM) may contribute to optimizing the efficacy and safety of antifungal therapy because of the large variability in drug pharmacokinetics. Rapid, sensitive, and selective laboratory methods are needed for efficient TDM. Quantification of several antifungals in a single analytical run may best fulfill these requirements. We therefore developed a multiplex ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method requiring 100 μl of plasma for simultaneous quantification within 7 min of fluconazole, itraconazole, hydroxyitraconazole, posaconazole, voriconazole, voriconazole-N-oxide, caspofungin, and anidulafungin. Protein precipitation with acetonitrile was used in a single extraction procedure for eight analytes. After reverse-phase chromatographic separation, antifungals were quantified by electrospray ionization-triple-quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. Deuterated isotopic compounds of azole antifungals were used as internal standards. The method was validated based on FDA recommendations, including assessment of extraction yields, matrix effect variability (<9.2%), and analytical recovery (80.1 to 107%). The method is sensitive (lower limits of azole quantification, 0.01 to 0.1 μg/ml; those of echinocandin quantification, 0.06 to 0.1 μg/ml), accurate (intra- and interassay biases of -9.9 to +5% and -4.0 to +8.8%, respectively), and precise (intra- and interassay coefficients of variation of 1.2 to 11.1% and 1.2 to 8.9%, respectively) over clinical concentration ranges (upper limits of quantification, 5 to 50 μg/ml). Thus, we developed a simple, rapid, and robust multiplex UPLC-MS/MS assay for simultaneous quantification of plasma concentrations of six antifungals and two metabolites. This offers, by optimized and cost-effective lab resource utilization, an efficient tool for daily routine TDM aimed at maximizing the real-time efficacy and safety of different recommended single-drug antifungal regimens and combination salvage therapies, as well as a tool for clinical research.

Variable viral clearance despite adequate ganciclovir plasma levels during valganciclovir treatment for cytomegalovirus disease in D+/R- transplant recipients.

ABSTRACT: BACKGROUND: Valganciclovir, the oral prodrug of ganciclovir, has been demonstrated equivalent to iv ganciclovir for CMV disease treatment in solid organ transplant recipients. Variability in ganciclovir exposure achieved with valganciclovir could be implicated as a contributing factor for explaining variations in the therapeutic response. This prospective observational study aimed to correlate clinical and cytomegalovirus (CMV) viral load response (DNAemia) with ganciclovir plasma concentrations in patients treated with valganciclovir for CMV infection/disease. METHODS: Seven CMV D+/R- transplant recipients (4 kidney, 2 liver and 1 heart) were treated with valganciclovir (initial dose was 900-1800 mg/day for 3-6.5 weeks, followed by 450-900 mg/day for 2-9 weeks). DNAemia was monitored by real time quantitative PCR and ganciclovir plasma concentration was measured at trough (Ctrough) and 3 h after drug administration (C3h) by HPLC. RESULTS: Four patients presented with CMV syndrome, two had CMV tissue-invasive disease after prophylaxis discontinuation, and one liver recipient was treated pre-emptively for asymptomatic rising CMV viral load 5 weeks post-transplantation in the absence of prophylaxis. CMV DNAemia decreased during the first week of treatment in all recipients except in one patient (median decrease: -1.2 log copies/mL, range: -1.8 to 0) despite satisfactory ganciclovir exposure (AUC0-12 = 48 mg.h/L, range for the 7 patients: 40-118 mg.h/L). Viral clearance was obtained in five patients after a median of time of 34 days (range: 28-82 days). Two patients had recurrent CMV disease despite adequate ganciclovir exposure (65 mg.h/L, range: 44-118 mg.h/L). CONCLUSIONS: Valganciclovir treatment for CMV infection/disease in D+/R- transplant recipients can thus result in variable viral clearance despite adequate ganciclovir plasma concentrations, probably correlating inversely with anti-CMV immune responses after primary infection.

Determination of human plasma levels of levo-alpha-acetylmethadol and its metabolites by gas chromatography-mass spectrometry.

A gas chromatography-mass spectrometry (GC-MS) method is presented which allows the simultaneous determination of the plasma concentrations of the levo-alpha-acetylmethadol (LAAM) and of its active metabolites (NorLAAM and DiNorLAAM), after derivatization with the reagent trifluoroacetic anhydride (TFAA). No interferences from endogenous compounds were observed following the extraction of plasma samples from 11 different human subjects. The standard curves were linear over a working range of 5-200ng/ml for the three compounds. Recoveries measured at three concentrations ranged from 47 to 67% for LAAM, from 50 to 69% for NorLAAM and from 28 to 50% for DiNorLAAM. Intra- and interday coefficients of variation determined at three concentrations ranged from 5 to 13% for LAAM, from 3 to 9% for NorLAAM and from 5 to 13% for DiNorLAAM. The limits of quantitation of the method were found to be 4ng/ml for the three compounds. No interference was noted from methadone. This sensitive and specific analytical method could be useful for assessing the in vivo relationship between LAAM's blood levels, clinical efficacy and/or cardiotoxicity