Zuzana Havlínová

Nitrite in exhaled breath condensate as a marker of nitrossative stress in the airways of patients with asthma, COPD, and idiopathic pulmonary fibrosis

Background: Nitrite and nitrate are exhaled in droplets of an aerosol during breathing and can be assayed in the exhaled breath condensate (EBC) as markers of nitrossative stress in the airways of patients with asthma, COPD, and idiopathic pulmonary fibrosis (IPF). Subjects and methods: Using HPLC with fluorescence detection, nitrite and nitrate were assayed in EBC of 14 atopic patients with mild‐to‐moderate stable asthma, 18 atopic asthmatics with exacerbation, 14 COPD patients without exacerbation, 18 patients with exacerbated COPD, 13 patients with active IPF, and in 29 healthy subjects. Results: The geometric mean [exp(mean±SD)] EBC concentrations of nitrite (micromol/l) in patients with asthma [5.1(2.1–12.3)], exacerbation of asthma [5.1(2.8–9.6)], exacerbation of COPD [5.3(3.2–8.7)], and with IPF [5.5(2.9–10.2)] were higher (P<0.05) compared with those of healthy subjects [2.9(1.6–5.3)] and patients with stable COPD [3.0(1.3–6.7)]. Nitrite concentration increased with decreased lung function of patients with asthma (rs=−0.31, P<0.02). Presumably owing to the contamination of the EBC sample with nitrate during collection, nitrate levels were highly variable among healthy subjects and higher compared with all groups of patients. Conclusion: EBC nitrite is a suitable marker of nitrossative stress in adult patients with lung diseases but cannot differentiate controlled and exacerbated asthma. Further improvements to the methods of EBC collection and sample handling are warranted. J. Clin. Lab. Anal. 24:317–322, 2010.

Analysis of Single-breath Profiles of Exhaled Nitric Oxide in Children with Allergy and Asthma: Guideline-derived Plateau Concentrations Compared to Results of Automatic Evaluation by Two Analyzers

Current guidelines recommend the single-breath measurement of fractional concentration of exhaled nitric oxide (FENO) at the expiratory flow rate of 50 mL/s as a gold standard. The time profile of exhaled FENO consists of a washout phase followed by a plateau phase with a stable concentration. This study performed measurements of FENO using a chemiluminescence analyzer Ecomedics CLD88sp and an electrochemical monitor NIOX MINO in 82 children and adolescents (44 males) from 4.9 to 18.7 years of age with corticosteroid-treated allergic rhinitis (N = 58) and/or asthma (N = 59). Duration of exhalation was 6 seconds for children less than 12 years of age and 10 seconds for older children. The first aim was to compare the evaluation of FENO-time profiles from Ecomedics by its software in fixed intervals of 7 to 10 seconds (older children) and 2 to 4 seconds (younger children) since the start of exhalation (method A) with the guideline-based analysis of plateau concentrations at variable time intervals (method B). The second aim was to assess the between-analyzer agreement. In children over 12 years of age, the median ratio of FENO concentrations of 1.00 (95% CI: 0.99–1.02) indicated an excellent agreement between the methods A and B. Compared with NIOX MINO, the Ecomedics results were higher by 11% (95% CI: 1–22) (method A) and 14% (95% CI: 4–26) (method B), respectively. In children less than 12 years of age, the FENO concentrations obtained by the method B were 34% (95% CI: 21–48) higher and more reproducible (p < 0.02) compared to the method A. The Ecomedics results of the method A were 11% lower (95% CI: 2–20) than NIOX MINO concentrations while the method B gave 21% higher concentrations (95% CI: 9–35). We conclude that in children less than 12 years of age, the guideline-based analysis of FENO–time profiles from Ecomedics at variable times obtains FENO concentrations that are higher and more reproducible than those from the fixed interval of 2 to 4 seconds and higher than NIOX MINO concentrations obtained during a short exhalation (6 seconds). The Ecomedics FENO concentrations of children more than 12 years of age calculated in the interval of 7 to 10 seconds represent plateau values and agree well with NIOX MINO results obtained during a standard 10-second exhalation.

HPLC determination of arginases inhibitor N-(ω)-hydroxy-nor-L-arginine using core-shell particle column and LC-MS/MS identification of principal metabolite in rat plasma.

For the purpose of in vivo pharmacokinetic studies, an HPLC method was developed and validated for the quantification of N-(ω)-hydroxy-nor-L-arginine, L-arginine and N-(ω)-ethyl-L-arginine (internal standard) in rat plasma. Sample processing involved a solid-phase extraction on the Waters MCX cartridges and on-line pre-column derivatization of the analytes with o-phthaldialdehyde and 3-mercaptopropionic acid. Separation of the derivatives was carried out on a core-shell Kinetex C18 column in a gradient elution mode with a mobile phase consisting of methanol and water (pH=3.00 adjusted with formic acid). Fluorimetric detection with the excitation/emission wavelengths of 235/450 nm was used. The method was validated according to the FDA guidelines and applied to pilot pharmacokinetic experiments. An unknown metabolite was extracted from the plasma of Wistar rats after a single bolus of N-(ω)-hydroxy-nor-L-arginine (i.v. 10 mg kg(-1)). The metabolite was identified as nor-L-arginine using mass spectrometry. Validated method was successfully used for pilot pharmacokinetic experiment on rats.

Early changes in L-arginine-nitric oxide metabolic pathways in response to the whole-body gamma irradiation of rats

Abstract Purpose: Nitric oxide (NO), a reactive radical, is formed in higher amounts from L-arginine by inducible NO synthase (iNOS) during early response to ionizing radiation presumably as a part of signal transduction pathways. This study investigated the changes in L-arginine-NO metabolic pathways within a 24-hour period after whole-body gamma irradiation of rats with the range of low to supra-lethal doses. Materials and methods: Young adult female Wistar rats received either 0–50 Gy whole-body irradiation or an intraperitoneal injection of bacterial lipopolysaccharide (LPS, 10 mg/kg). Exhaled NO was monitored using chemiluminiscence, nitrite + nitrate (NOx) and L-arginine were assayed by high-performance liquid chromatography, and expression of iNOS was determined using Western blot. Results: Irradiation resulted in a dose-dependent increase of plasma NOx to maximum levels which were 4-fold higher compared to controls (p < 0.001). The NOx levels increased less in the bronchoalveolar lavage fluid (BAL) (1.7-fold, p < 0.001) and liver homogenate (2.5-fold, p < 0.05), respectively, and were dose-independent. Exhaled NO, lung NOx, plasma and BAL L-arginine, and the expression of iNOS in lung and liver tissues of irradiated rats and controls were similar. LPS caused a considerable increase (p < 0.001) in exhaled NO (61-fold), NOx levels (plasma 34-fold, BAL 6-fold, lung 5-fold, liver 4-fold), and in iNOS expression, respectively. Conclusion: In contrast to the LPS treatment of rats, the radiation-induced changes in L-arginine-NO metabolic pathways are modest, particularly in the airways and lungs. Noninvasive measurement of exhaled NO within a 24-h period following the exposure of rats to ionizing radiation has no value for biodosimetry.

IL-1 receptor blockade alleviates endotoxin-mediated impairment of renal drug excretory functions in rats

The aim of our study was to investigate whether two potent anti-inflammatory agents, dexamethasone and anakinra, an IL-1 receptor antagonist, may influence acute kidney injury (AKI) and associated drug excretory functions during endotoxemia (LPS) in rats. Ten hours after LPS administration, untreated endotoxemic rats developed typical symptoms of AKI, with reduced GFR, impaired tubular excretion of urea and sodium, and decreased urinary excretion of azithromycin, an anionic substrate for multidrug resistance-transporting proteins. Administration of both immunosuppressants attenuated the inflammatory response, liver damage, AKI, and increased renal clearance of azithromycin mainly by restoration of GFR, without significant influence on its tubular secretion. The lack of such an effect was related to the differential effect of both agents on the renal expression of individual drug transporters. Only dexamethasone increased the urinary clearance of bile acids, in accordance with the reduction of the apical transporter (Asbt) for their tubular reabsorption. In summary, our data demonstrated the potency of both agents used for the prevention of AKI, imposed by endotoxins, and for the restoration of renal drug elimination, mainly by the improvement of GFR. The influence of both drugs on altered tubular functions and the expression of drug transporters was differential, emphasizing the necessity of knowledge of transporting pathways for individual drugs applied during sepsis. The effect of anakinra suggests a significant contribution of IL-1 signaling to the pathogenesis of LPS-induced AKI.

Single- and multiple-dose pharmacokinetics of arginase inhibitor Nω-hydroxy-nor-L-arginine, and its effect on plasma amino acids concentrations in Wistar rats.

Arginase inhibitor Nω-hydroxy-nor-L-arginine (nor-NOHA) augments synthesis of nitric oxide (NO) exerting therapeutic effects in rodent models for cardiovascular and airway diseases. This study examined single- and multiple-dose pharmacokinetics and effects of nor-NOHA on plasma amino acids in Wistar rats. Animals were administered 30 mg/kg nor-NOHA in a single bolus intravenous (i.v.) or intraperitoneal (i.p.) injection, or five once-daily i.p. injections at the same dose, or vehicle. Nor-NOHA and amino acids were assayed in blood plasma by high-performance liquid chromatography. After a bolus i.v. injection, the elimination of nor-NOHA was rapid (the mean residence time was 12.5 min). The area under the concentration-time curve and maximum concentration were higher by 17% and 31%, respectively, after the fifth as compared to the first i.p. injection. A shift in arginine utilization towards the synthesis of NO was indicated by elevated citrulline-to-ornithine and citrulline-to-arginine ratios. No changes in plasma arginine were observed. Increased glutamine concentrations might indicate an alternative detoxification pathway for ammonia due to inhibition of hepatic arginase. In conclusion, pharmacokinetic data of the present study can guide rational dosing of nor-NOHA in future studies. Limitations of the strategy of NO modulation via arginase inhibition should be further explored.

Comparative pharmacokinetics of Nω-hydroxy-nor-L-arginine, an arginase inhibitor, after single-dose intravenous, intraperitoneal and intratracheal administration to brown Norway rats

Abstract 1.  Rodent studies have documented that Nω-hydroxy-nor-L-arginine (nor-NOHA), an arginase inhibitor, has therapeutic potential in the treatment of cardiovascular and obstructive airway diseases. However, its bioavailability and pharmacokinetics have not been described so far. 2.  Anesthetized brown Norway rats were administered single doses of nor-NOHA (10, 30 or 90 mg/kg) intravenously (i.v.), intraperitonealy (i.p.) or via intratracheal (i.t.) instillation of aerosol. Plasma nor-NOHA was assayed using a validated HPLC method. 3.  Upon i.v. administration, the mean concentration showed a biphasic decline and its value dropped below 10% of the maximum after 20 min. The pharmacokinetics were linear with the total and inter-compartmental clearances of 33 and 17 mL/min/kg, central and peripheral volumes of distribution of 0.19 and 0.43 L/kg and terminal half-life of 30 min. 4.  The average absolute bioavailability of nor-NOHA after i.p. and i.t. delivery was 98% and 53%, respectively. The absorption from the airways was rate-limiting and its extent decreased with the dose. 5.  In conclusion, nor-NOHA is rapidly cleared from the plasma in concordance with the short time window of its in vivo inhibitory activity reported in the literature. I.t. instillation of aerosol for topical effects of nor-NOHA in the airways is characterized with significant systemic availability.

Alveolar concentration and bronchial flux of nitric oxide: two linear modeling methods evaluated in children and adolescents with allergic rhinitis and atopic asthma.

Alveolar concentration (CANO) and bronchial flux (JawNO) of nitric oxide (NO) characterize the contributions of peripheral and proximal airways to exhaled NO. Both parameters can be estimated using a two‐compartment model if the fraction of NO in orally exhaled air (FENO) is measured at multiple constant expiratory flow rates (V). The aim of this study was to evaluate how departures from linearity influence the estimates of CANO and JawNO obtained with the help of linear regression analysis of the relationships between FENO and 1/V (method P), and between the NO output (VNO = FENO × V) and V (method T). Furthermore, differences between patients with atopic asthma (AA) and allergic rhinitis (AR) and between methods P and T were assessed.