1380 Cristina Maccallini1 Mauro Di Matteo1 Alessandra Ammazzalorso1 Alessandra D’Angelo1 Barbara De Filippis1 Sara Di Silvestre2 Marialuigia Fantacuzzi1 Letizia Giampietro1 Assunta Pandolfi2 Rosa Amoroso1 ∗ 1 Dipartimento
di Farmacia, Universita` degli Studi “G. d’Annunzio”, Chieti, Italy 2 Dipartimento di Scienze Sperimentali e Cliniche, Centro Scienze dell’Invecchiamento, Ce.S.I.,‘Fondazione Universita` G. d’Annunzio’, Universita` degli Studi “G. d’Annunzio”, Chieti, Italy Received January 20, 2014 Revised March 13, 2014 Accepted March 19, 2014
J. Sep. Sci. 2014, 37, 1380–1385
Research Article
Reversed-phase high-performance liquid chromatography method with fluorescence detection to screen nitric oxide synthases inhibitors Nitric oxide synthase (NOS) inhibitors are potential drug candidates due to the critical role of an excessive production of nitric oxide in a range of diseases. At present, the radiometric detection of L-[3 H]-citrulline produced from L -[3 H]-arginine during the enzymatic reaction is one of the most accepted methods to assess the in vitro activity of NOS inhibitors. Here we report a fast, easy, and cheap reversed-phase high-performance liquid chromatography method with fluorescence detection, based on the precolumn derivatization of L-citrulline with ophthaldialdehyde/N-acetyl cysteine, for the in vitro screening of NOS inhibitors. To evaluate enzyme inhibition by the developed method, N-[3-(aminomethyl)benzyl]acetamidine, a potent and selective inhibitor of inducible NOS, was used as a test compound. The half maximal inhibitory concentration obtained was comparable to that derived by the well-established radiometric assay. Keywords: L-Citrulline / Fluorescence detection / High-performance liquid chromatography / Inhibitors / Nitric oxide synthases DOI 10.1002/jssc.201400059
1 Introduction Nitric oxide synthases (NOS) are a family of enzymes deputed to the biosynthesis of nitric oxide (NO) and L-citrulline (Citr) from L-arginine (Arg), in the presence of O2 , nicotinamide adenine dinucleotide phosphate (NADPH) and other cofactors (Scheme 1). In mammals, three isoforms of NOS have been recognized: the constitutive endothelial (eNOS) and neuronal (nNOS) ones, predominantly expressed in the vascular endothelium and in the nervous system, respectively, and the inducible (iNOS) isoform, that generates high levels of NO, modulating inflammation through multiple pathways and playing an important role in the regulation of immune reactions [1–4]. The unregulated overproduction
Correspondence: Dr. Cristina Maccallini, Dipartimento di Farmacia, Universita` degli Studi “G. d’Annunzio”, Via dei Vestini 31, 66100 Chieti, Italy E-mail: [email protected] Fax: +39 8713554681
Abbreviations: Arg, L-arginine; BH4 , tetrahydrobiopterin; CaM, calmodulin; Citr, L-citrulline; DTT, D,L-dithiothreitol; eNOS, endothelial nitric oxide synthase; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; HbO2 , oxyhemoglobin; HEPES, 2-[4-(2-hydroxyethyl)piperazin-1yl]ethanesulfonic acid; IC50 , half maximal inhibitory concentration; iNOS, inducible nitric oxide synthase; NAC, N-acetyl cysteine; NADPH, nicotinamide adenine dinucleotide phosphate; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; NOS, nitric oxide synthases; OPA, o-phthaldialdehyde
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of NO by nNOS and iNOS seems to be involved in the pathophysiology of several human diseases, such as neurodegeneration, stroke, asthma, arthritis, multiple sclerosis, colitis, psoriasis, tumor development, transplant rejection and septic shock [5–9]. For this reason the selective inhibition of nNOS or iNOS appears to be a therapeutic tool, and different types of NOS inhibitors have been described in the past few years [10–18]. The biological activity of a NOS inhibitor is expressed by its half maximal inhibitory concentration (IC50 ), which can be assessed by different enzymatic assays. In general, the residual NOS activity after incubation with the molecule, is evaluated by measuring NO or Citr levels, as they are the products formed during the enzymatic reaction (Scheme 1) [19]. Nitrite and nitrate ions (NOx ) are the product of NO oxidation in aqueous aerobic solvents, and their detection represents an index of the NO production trend. Nitrite ions (NO2 − ) can be spectrophotometrically detected by observing the magenta-colored azodye that is formed after reaction with the Griess reagent [20, 21]. Anyway a problem with this assay is that nitrate ions must be first reduced to NO2 − with nitrate reductase or a copper-plated cadmium column. Moreover, possible NOx sample contamination could occur, with a consequent overestimation of NO sample contents [22, 23]. NOS activity can be spectrophotometrically measured also by exploiting the NO reaction with oxyhemoglobin (HbO2 ) to form ∗ Additional corresponding author: Professor Rosa Amoroso, E-mail: [email protected] Colour Online: See the article online to view Figs. 3–5 in colour.
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2 Materials and methods 2.1 Chemicals and reagents
Scheme 1. Reaction catalyzed by NOS.
All chemicals, HPLC grade solvents, and DOWEX 50-W X 8– 400 ion exchange resin, were supplied from Sigma–Aldrich (St. Louis, MO, USA). 1400W was synthesized in our lab [28]. Recombinant murine iNOS was purchased from Bertin Pharma (Montigny-le-Bretonneux, France). L-[H]3 Arginine and scintillation fluid ULTIMA GOLD were purchased from Perkin Elmer (MA, USA). HPLC-grade water was obtained by passage through an Elga Purelab water purification system (Elga Labwater, UK). A centrifuge EBA21 (Hettich, Germany) was used. A liquid scintillation analyzer, model TRI-CARB 2100 TR from Perkin Elmer was used. 2.2 Standard solutions
Figure 1. Chemical structure of N-[3-(aminomethyl)benzyl]acet amidine (W1400).
methemoglobin which absorbs at 401 nm [24]. On one hand, the hemoglobin capture of NO offers the advantage of a kinetic detection of NO but, on the other, a problem with this assay is the reaction between HbO2 and interfering species like free peroxynitrite ions [25]. The citrulline assay is a sensitive, discontinuous radiometric assay, assessing the amount of L-[3 H]citrulline or L-[14 C]citrulline formed by the conversion of L-[3 H]arginine or L-[14 C]arginine, respectively [26]. Critical aspects are the purity of the starting radiolabeled arginine, the pH of the cationic resin employed to separate the obtained radiolabeled citrulline, and the costs of materials [27]. Several HPLC methods have been reported for the determination of Citr levels in different biological fluids and tissues, often based on sample precolum derivatization with o-phthaldialdehyde (OPA) [28–31]. Anyway, to the best of our knowledge, none of the published methods has been applied to evaluate Citr produced by NOS during the in vitro enzymatic reaction in the presence of inhibitors. In view of this, we have developed an alternative, simple and sensitive chromatographic method to evaluate the enzymatic inhibition of NOS, based on the fluorescence detection of Citr derivatized with an o-phthalaldehyde/N-acetyl cysteine (OPA/NAC) reagent. To evaluate NOS inhibition, we used N[3-(aminomethyl)benzyl]acetamidine (1400W, Fig. 1), which is the most selective inhibitor of purified human iNOS reported to date (5000- and 200-fold more potent against purified human iNOS than eNOS and nNOS, respectively) [32]. Results obtained by the developed method are compared to those obtained by the well established citrulline radiometric assay. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The L-arginine and L-citrulline stock solutions (100 g/mL) were prepared in HPLC-grade water and then diluted with the appropriate volume of HPLC grade water, to obtain standard solutions in the concentration range of 0.001–100 M. Validation standard solutions in the concentration range 0.001–10 M were obtained spiking enzymatic assay mixture with Citr stock solutions. Stock solution of methanolic OPA contained 0.256 g OPA in 50 mL methanol. iNOS stock solution (100 g/mL) was prepared in 40 mM HEPES buffer pH = 7.4 and stored at –80⬚C. Radiolabeled L-arginine stock solutions (s.s, 40–70 Ci/mL) were stored at 4⬚C and used without further purification. 2.3 Methods 2.3.1 Enzymatic assay conditions To perform the enzymatic assay, 10 L of iNOS stock solution was added to 80 L of HEPES buffer pH = 7.4, containing 0.1 mM CaCl2 , 1 mM DTT, 0.5 mg/mL BSA, 10 M FMN, 10 M FAD, 30 M BH4 , 10 g/mL CaM, and 11 M Larginine. For the citrulline radiometric detection, 2 L of L-[H]3 arginine s.s. were added. To measure enzyme inhibition, a 10 L solution of 1400W (0.001–100 M) was added to the enzyme assay solution, followed by a pre-incubation time of 15 min at room temperature. The reaction was initiated by addition of 10 L of NADPH 7.5 mM and was carried out at 37⬚C for 20 min. Then it was stopped by following Method a) for the citrulline fluorimetric detection or Method b) for the radiometric detection. Method a): 500 L of ice-cold CH3 CN was added to the reaction and the mixture was brought to dryness under vacuum and eventually stored at –20⬚C, before the fluorescence derivatization. Method b): 500 L of ice-cold HEPES Na+ pH = 6.0 were added to the reaction, and L-[H]3 citrulline was www.jss-journal.com
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separated by DOWEX, which was washed with 1.2 mL of water. A 500 L aliquot of the eluate was added to 4 mL of scintillation fluid and the radioactivity was counted in a liquid scintillation counter. All assays were performed in triplicate. 2.3.2 Fluorescence derivatization The OPA/NAC reagent was prepared by mixing for 90 min, 5 mL of methanolic OPA solution and 20 mL of 0.2 M borate buffer containing 0.1 g of NAC. Final pH = 9.3 ± 0.05; the molar ratios of OPA to NAC was 1:3. The OPA/NAC solution was stored at 4⬚C and saved for no longer than seven days. Immediately before the HPLC analysis, the enzymatic assay residue was dissolved in 600 L of HPLC-grade water, and centrifuged at 6000 rpm for 35 min. Then 190 L of supernatant or amino acid stock solution and 60 L of OPA/NAC solution were stirred for 5 min and injected into column. 2.3.3 Apparatus and chromatographic conditions HPLC analyses were performed using a Waters (Milford, MA, USA) system composed of a P600 model pump, a 2475 multi-fluorescence detector, and a 7725i model sample injector (Rheodyne, Cotati, CA, USA) equipped with a 5 L loop. The analyses were performed on an XTerra MS C8 column (250 × 4.6 mm id, 5 m particle size; Waters), equipped with an XTerra MS C8 guard column (Waters). A column thermostat oven module Igloo-Cil (Cil Cluzeau Info Labo, France) was used. Chromatograms were recorded on a Fujitsu Siemens Esprimo computer and data were processed by the Empower Pro software (Waters). The column was eluted at a flow rate of 0.7 mL/min with linear gradients of buffers A (5% in CH3 CN in 15 mM sodium borate with 0.1% v/v trifluoroacetic acid, pH = 9.4) and B (50% in CH3 CN in 8 mM sodium borate with 0.1% v/v trifluoroacetic acid, pH = 9.4). The solvent gradient was: 0–20% B at 0–10 min, increasing B to 25% at 10–15 min, then to 40% at 15–20 min and to 70% at 20–28 min. This composition was maintained until t = 35 min, before being reduced to the initial 0% B composition. The mobile phase was prepared daily, filtered through a 0.45 m WTP membrane (Whatman, Maidstone, UK), sonicated and degassed before use. Column temperature was kept constant at 20⬚C. The injection volume was 5 L. The fluorescence intensity in the column eluate was monitored at 439 nm (emission) with excitation at 335 nm. 2.4 Method evaluation 2.4.1 Linearity, matrix effect study, LOD and LOQ Linearity was evaluated by the construction of calibration curves of solutions containing Citr in the range of 0.001– 100 M. The regression equation is y = 18303x + 403697. To study sample matrix effect, calibration curves of Citr validation standards in the concentration range of 0.001– C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
10 M were constructed. The regression equation for the obtained calibration curves is y = 17200x + 1419140. Proportional bias was evaluated as the ratio between validation and standard calibration curves slopes, while constant bias was determined as (validation curve intercept – calibration curve intercept) / validation curve slope. The LOD value was calculated from the residual SD of the regression line () of the analytical curve and its slope, according to the equations LOD = 3.3 (/S) and LOQ = 10(/S). 2.4.2 Accuracy and precision Accuracy was determined by analyzing in triplicate validation standards spiked with 0.5, 1 and 5 M Citr. Each sample was analyzed in three separate runs. Accuracy was calculated as: 100 × (overall measured concentration – unspiked native concentration)/ added concentration. The precision of the method was calculated by three validation standard solutions at 0.5, 1 and 5 M Citr. The intraday precision was calculated by six consecutive injections of each solution in one day, while the inter-day precision was evaluated by three injections of the same solution kept at 4⬚C, on three consecutive days. 2.4.3 Stability Stability was checked by injecting after one and three days 1 M Citr-derivatized sample solutions stored at 4⬚C. Peak areas were compared to those of freshly prepared samples. 2.4.4 Statistical analysis Statistical analysis of the data was achieved by using Prism 5 (GraphPad Software, La Jolla, CA, USA). The findings were regarded as significant if P values were < 0.05.
3 Results and discussion 3.1 HPLC conditions NOS catalyze the conversion of 1 equiv. of L-arginine into 1 equiv. of NO and L-citrulline, so the RPLC fluorescence detection of Citr levels represents an easy evaluation of the enzymatic activity. Our first goal was to optimize HPLC conditions to obtain a complete resolution of the OPA/NAC derivatized Citr in the enzymatic assay mixture. L-Citrulline and L-arginine standard solutions were used to set up the method, and different mobile phase compositions and ratios were attempted: phosphate and acetate buffers (from pH = 2 to 7.5) provided poor sensitivity and/or resolution, while sodium borate (pH = 9.4, 15 mM) gave the best results. The addition of 0.1% v/v of TFA ameliorated the peak shape. We adopted CH3 CN as the organic modifier, and as isocratic elution did not afford resolution of derivatized Arg and Citr, we decided to apply a mild gradient of two mobile phases A and B, www.jss-journal.com
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Table 1. Accuracy of determination of L-citrulline in NOS mixture assay (n = 9)
Citrulline added (M)
Measured (M)
Recovery (%)
0.1 1 5
0.113 1.078 4.921
97.8 106.3 98.4
Table 2. Intra- and inter-day precision of determination of L-citrulline in NOS mixture assay
Intra-day precision (n = 6)% RSD Inter-day precision (n = 9)% RSD
0.1
1
5
2.05 3.40
1.24 2.80
0.97 2.94
adopted method. The theoretical plate number for OPA/NAC derivatized amino acids was more than 5000. 3.3 Linearity, LOD and LOQ
Figure 2. Chromatograms of derivatized L-citrulline (A) and Larginine (B) standard solutions.
Figure 3. Chromatogram of a derivatized enzymatic assay mixture in absence of inhibitors (control).
containing, respectively, 5 and 50% of CH3 CN in sodium borate with 0.1% v/v of TFA.
3.2 System suitability Figure 2 shows typical chromatograms of amino acid calibration standard solutions, and Fig. 3 represents a chromatogram of an enzymatic assay mixture in absence of inhibitors (control). The retention times of derivatized Citr and Arg were 15 and 17 min, respectively, and the absence of overlapping or interfering signals supports the selectivity of the
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Linearity was evaluated for derivatized Citr standard solutions in the range of 0.001–100 M. Straight regression line with correlation coefficient of 0.999 was obtained. The Ftest applied for calibration curve and data was 2118 and provides conclusive evidence of a linear relationship between concentration and instrumental response. The LOQ was 183 nmol/L, while a value of 60 nmol/L was calculated as LOD. To evaluate bias derived from sample matrix effects, Citr spiked enzymatic assay mixtures in the range of 0.001–10 M were used to obtain calibration curves. The correlation coefficient of the obtained regression line was 0.993. Proportional bias was found, due to the different slopes in standard and validation calibration curves and it was 0.940, while constant bias was 55.480. The LOD obtained from validation standard curve was 85 nmol/L, and the LOQ was 258 nmol/L. 3.4 Accuracy and precision In order to measure accuracy and precision of the method, triplicate of 0.5, 1 and 5 M Citr validation standard solutions were analyzed. Recovery results are summarized in Table 1. Intraday and interday precision are expressed as the percentage RSD% for Citr peak area and data are reported in Table 2.
3.5 Stability Derivatized sample solutions proved to be stable for three days at least at 4⬚C. Indeed, recovery was 106.7 and 105% after one and three days, respectively.
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Figure 5. Radiometric evaluation of the dose-dependent inhibition of iNOS by 1400W. The results are from three independent experiments and are expressed as means ± standard deviations.
were stopped with cold HEPES sodium, then purified on ion exchange resins and finally added scintillation fluid. The obtained IC50 curve is shown in Fig. 5, and the extrapolated IC50 value was 0.09 M, similar to that obtained by the fluorescence detection.
4 Concluding remarks Figure 4. (A) Peak area values of derivatized L-citrulline in the presence of different 1400W concentrations in comparison with the absence of inhibitor (CTRL) and blank (BLK). The results are from three independent experiments and are expressed as means ± standard deviations. (B) Fluorimetric RPLC evaluation of dosedependent inhibition of iNOS by 1400W. The results are from three independent experiments and are expressed as means ± standard deviations.
3.6 Evaluation of 1400W IC50 To assess the suitability of our chromatographic method in the evaluation of NOS inhibitors potency, 1400W was used as a test compound. In typical enzymatic reaction conditions, different 1400W concentrations were incubated with iNOS. After incubation, reactions were stopped with ice-cold CH3 CN, dried and if necessary, stored at –20⬚C. Before the analysis, the solid residue was suspended in HPLC-grade water, derivatized with OPA/NAC reagent and analyzed. Figure 4A shows the results of the fluorescence detection, after RPLC separation, of derivatized L-citrulline as the product of iNOS activity in the absence (control) of inhibitor and in the presence of different of W1400 concentrations; and Fig. 4B shows the obtained dose-dependent inhibition of the enzyme by 1400W. An IC50 value of 0.08 M was calculated, comparable to the published data [13]. For comparison, we also evaluated IC50 by the well established radiometric assay, which is the most employed method in the evaluation of NOSs inhibitors potency. After incubation, reactions C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The selective inhibition of both neuronal and/or inducible NOS could be a new therapeutic opportunity in different diseases, so precise, reproducible, fast, and economic screening of new inhibitors is worthwhile. Here we have demonstrated a convenient method to evaluate in vitro NOS activity, alternative to the most used radiometric assay. In particular, we avoid the presence of any radioactive materials, expensive scintillation fluid and time-consuming chromatographic purification steps. The proposed derivatization step can be totally automated using an autosampler and the obtained solutions proved to be stable at 4⬚C for three days at least. So this successful RP-HPLC method with Citr fluorescence detection can be used as a reliable procedure for screening the biological activity of NOS inhibitors. This study was supported by University “G. d’Annunzio” of Chieti local grants. The authors have declared no conflict of interest.
5 References [1] Lirk, P., Hoffmann, G., Rieder, J., Curr. Drug Targets Inflamm. Allergy 2002, 1, 89–108. [2] Feng, C., Coord. Chem. Rev. 2012, 256, 393–411. [3] Li, H., Poulos, T. L., J. Inorg. Biochem. 2005, 99, 293–305. [4] Santolini, J., J. Inorg. Biochem. 2011, 105, 127–141.
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[5] Knott, A. B., Bossy-Wetzel, E., Antioxid. Redox Signal 2009, 11, 541–553. [6] Hooper, W. C., Curr. Pharm. Des. 2004, 10, 923–927. [7] Korhonen, R., Lahti, A., Kankaanranta, H., Moilanen, E., Curr. Drug Targets Inflamm. Allergy 2005, 4, 471–479.
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[10] Tinker, A. C., Wallace, A. V., Curr. Top. Med. Chem. 2006, 6, 77–79. [11] Zhou, L., Zhu, D. Y., Nitric Oxide 2009, 20, 223–230. [12] Muscara, M. N., Wallace, J. L., Am. J. Physiol. 1999, 276, 1313–1316. [13] Maccallini, C., Patruno, A., Besker, N., Ali, J. I., Ammazzalorso, A., De Filippis, B., Franceschelli, S., Giampietro, L., Pesce, M., Reale, M., Tricca, M. L., Re, N., Felaco, M., Amoroso, R., J. Med. Chem. 2009, 52, 1481–1485. [14] Maccallini, C., Patruno, A., Lannutti, F., Ammazzalorso, A., De Filippis, B., Fantacuzzi, M., Franceschelli, S., Giampietro, L., Masella, S., Felaco, M., Re, N., Amoroso, R., Bioorg. Med. Chem. Lett. 2010, 20, 6495–6499. [15] Fantacuzzi, M., Maccallini, C., Lannutti, F., Patruno, A., Masella, S., Pesce, M., Speranza, L., Ammazzalorso, A., De Filippis, B., Giampietro, L., Re, N., Amoroso, R., ChemMedChem 2011, 6, 1203–1206. [16] Maccallini, C., Patruno, A., Ammazzalorso, A., De Filippis, B., Fantacuzzi, M., Franceschelli, S., Giampietro, L., Masella, S., Tricca, M. L., Amoroso, R., Med. Chem. 2012, 8, 991–995. [17] Patruno, A., Franceschelli, S., Pesce, M., Maccallini, C., Fantacuzzi, M., Speranza, L., Ferrone, A., De Lutiis, M. A.,
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[22] Ishibashi, T., Himeno, M., Imaizumi, N., Maejima, K., Nakano, S., Uchida, K., Yoshida, J., Nishio, M., Nitric Oxide 2000, 4, 516–525. [23] Hunter, R. A., Storm, W. L., Coneski, P. N., Schoenfisch, M. H., Anal. Chem. 2013, 85, 1957–1963. [24] Hevel, J. M., Marletta, M. A., Meth. Enzymol 1994, 233, 250–258. [25] Schmidt, K., Klatt, P., Mayer, B., Biochem. J. 1994, 301, 645–647. [26] Bredt, D. S., Snyder, H., Proc. Natl. Acad. Sci. USA 1990, 87, 682–685. [27] Pandolfi, A., Grilli, A., Cilli, C., Patruno, A., Giaccari, A., Di Silvestre, S., De Lutiis, M. A., Pellegrini, G., Capani, F., Consoli, A., Felaco, M., J. Cell. Physiol. 2003, 196, 378– 385. [28] Abu-Soud, H. M., Presta, A., Mayer, B., Stuehr, D. J., Biochemisty 1997, 36, 10811–10816. [29] Mao, H., Chen, B., Wang, W., Zhuang, P., Zong, M., Xu, Z., Microchemical J. 2011, 97, 291–295. [30] Wu, G., Meininger, C. J., Meth. Enzymol. 2008, 440, 177– 189. [31] Carlberg, M., J. Neurosci. Meth. 1994, 52, 165–167. [32] Zhu, Y., Nikolic, D., Van Breemen, R. B., Silverman, R. B., J. Am. Chem. Soc. 2005, 127, 858–868.
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di Farmacia, Universita` degli Studi “G. d’Annunzio”, Chieti, Italy 2 Dipartimento di Scienze Sperimentali e Cliniche, Centro Scienze dell’Invecchiamento, Ce.S.I.,‘Fondazione Universita` G. d’Annunzio’, Universita` degli Studi “G. d’Annunzio”, Chieti, Italy Received January 20, 2014 Revised March 13, 2014 Accepted March 19, 2014
J. Sep. Sci. 2014, 37, 1380–1385
Research Article
Reversed-phase high-performance liquid chromatography method with fluorescence detection to screen nitric oxide synthases inhibitors Nitric oxide synthase (NOS) inhibitors are potential drug candidates due to the critical role of an excessive production of nitric oxide in a range of diseases. At present, the radiometric detection of L-[3 H]-citrulline produced from L -[3 H]-arginine during the enzymatic reaction is one of the most accepted methods to assess the in vitro activity of NOS inhibitors. Here we report a fast, easy, and cheap reversed-phase high-performance liquid chromatography method with fluorescence detection, based on the precolumn derivatization of L-citrulline with ophthaldialdehyde/N-acetyl cysteine, for the in vitro screening of NOS inhibitors. To evaluate enzyme inhibition by the developed method, N-[3-(aminomethyl)benzyl]acetamidine, a potent and selective inhibitor of inducible NOS, was used as a test compound. The half maximal inhibitory concentration obtained was comparable to that derived by the well-established radiometric assay. Keywords: L-Citrulline / Fluorescence detection / High-performance liquid chromatography / Inhibitors / Nitric oxide synthases DOI 10.1002/jssc.201400059
1 Introduction Nitric oxide synthases (NOS) are a family of enzymes deputed to the biosynthesis of nitric oxide (NO) and L-citrulline (Citr) from L-arginine (Arg), in the presence of O2 , nicotinamide adenine dinucleotide phosphate (NADPH) and other cofactors (Scheme 1). In mammals, three isoforms of NOS have been recognized: the constitutive endothelial (eNOS) and neuronal (nNOS) ones, predominantly expressed in the vascular endothelium and in the nervous system, respectively, and the inducible (iNOS) isoform, that generates high levels of NO, modulating inflammation through multiple pathways and playing an important role in the regulation of immune reactions [1–4]. The unregulated overproduction
Correspondence: Dr. Cristina Maccallini, Dipartimento di Farmacia, Universita` degli Studi “G. d’Annunzio”, Via dei Vestini 31, 66100 Chieti, Italy E-mail: [email protected] Fax: +39 8713554681
Abbreviations: Arg, L-arginine; BH4 , tetrahydrobiopterin; CaM, calmodulin; Citr, L-citrulline; DTT, D,L-dithiothreitol; eNOS, endothelial nitric oxide synthase; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; HbO2 , oxyhemoglobin; HEPES, 2-[4-(2-hydroxyethyl)piperazin-1yl]ethanesulfonic acid; IC50 , half maximal inhibitory concentration; iNOS, inducible nitric oxide synthase; NAC, N-acetyl cysteine; NADPH, nicotinamide adenine dinucleotide phosphate; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; NOS, nitric oxide synthases; OPA, o-phthaldialdehyde
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of NO by nNOS and iNOS seems to be involved in the pathophysiology of several human diseases, such as neurodegeneration, stroke, asthma, arthritis, multiple sclerosis, colitis, psoriasis, tumor development, transplant rejection and septic shock [5–9]. For this reason the selective inhibition of nNOS or iNOS appears to be a therapeutic tool, and different types of NOS inhibitors have been described in the past few years [10–18]. The biological activity of a NOS inhibitor is expressed by its half maximal inhibitory concentration (IC50 ), which can be assessed by different enzymatic assays. In general, the residual NOS activity after incubation with the molecule, is evaluated by measuring NO or Citr levels, as they are the products formed during the enzymatic reaction (Scheme 1) [19]. Nitrite and nitrate ions (NOx ) are the product of NO oxidation in aqueous aerobic solvents, and their detection represents an index of the NO production trend. Nitrite ions (NO2 − ) can be spectrophotometrically detected by observing the magenta-colored azodye that is formed after reaction with the Griess reagent [20, 21]. Anyway a problem with this assay is that nitrate ions must be first reduced to NO2 − with nitrate reductase or a copper-plated cadmium column. Moreover, possible NOx sample contamination could occur, with a consequent overestimation of NO sample contents [22, 23]. NOS activity can be spectrophotometrically measured also by exploiting the NO reaction with oxyhemoglobin (HbO2 ) to form ∗ Additional corresponding author: Professor Rosa Amoroso, E-mail: [email protected] Colour Online: See the article online to view Figs. 3–5 in colour.
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2 Materials and methods 2.1 Chemicals and reagents
Scheme 1. Reaction catalyzed by NOS.
All chemicals, HPLC grade solvents, and DOWEX 50-W X 8– 400 ion exchange resin, were supplied from Sigma–Aldrich (St. Louis, MO, USA). 1400W was synthesized in our lab [28]. Recombinant murine iNOS was purchased from Bertin Pharma (Montigny-le-Bretonneux, France). L-[H]3 Arginine and scintillation fluid ULTIMA GOLD were purchased from Perkin Elmer (MA, USA). HPLC-grade water was obtained by passage through an Elga Purelab water purification system (Elga Labwater, UK). A centrifuge EBA21 (Hettich, Germany) was used. A liquid scintillation analyzer, model TRI-CARB 2100 TR from Perkin Elmer was used. 2.2 Standard solutions
Figure 1. Chemical structure of N-[3-(aminomethyl)benzyl]acet amidine (W1400).
methemoglobin which absorbs at 401 nm [24]. On one hand, the hemoglobin capture of NO offers the advantage of a kinetic detection of NO but, on the other, a problem with this assay is the reaction between HbO2 and interfering species like free peroxynitrite ions [25]. The citrulline assay is a sensitive, discontinuous radiometric assay, assessing the amount of L-[3 H]citrulline or L-[14 C]citrulline formed by the conversion of L-[3 H]arginine or L-[14 C]arginine, respectively [26]. Critical aspects are the purity of the starting radiolabeled arginine, the pH of the cationic resin employed to separate the obtained radiolabeled citrulline, and the costs of materials [27]. Several HPLC methods have been reported for the determination of Citr levels in different biological fluids and tissues, often based on sample precolum derivatization with o-phthaldialdehyde (OPA) [28–31]. Anyway, to the best of our knowledge, none of the published methods has been applied to evaluate Citr produced by NOS during the in vitro enzymatic reaction in the presence of inhibitors. In view of this, we have developed an alternative, simple and sensitive chromatographic method to evaluate the enzymatic inhibition of NOS, based on the fluorescence detection of Citr derivatized with an o-phthalaldehyde/N-acetyl cysteine (OPA/NAC) reagent. To evaluate NOS inhibition, we used N[3-(aminomethyl)benzyl]acetamidine (1400W, Fig. 1), which is the most selective inhibitor of purified human iNOS reported to date (5000- and 200-fold more potent against purified human iNOS than eNOS and nNOS, respectively) [32]. Results obtained by the developed method are compared to those obtained by the well established citrulline radiometric assay. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The L-arginine and L-citrulline stock solutions (100 g/mL) were prepared in HPLC-grade water and then diluted with the appropriate volume of HPLC grade water, to obtain standard solutions in the concentration range of 0.001–100 M. Validation standard solutions in the concentration range 0.001–10 M were obtained spiking enzymatic assay mixture with Citr stock solutions. Stock solution of methanolic OPA contained 0.256 g OPA in 50 mL methanol. iNOS stock solution (100 g/mL) was prepared in 40 mM HEPES buffer pH = 7.4 and stored at –80⬚C. Radiolabeled L-arginine stock solutions (s.s, 40–70 Ci/mL) were stored at 4⬚C and used without further purification. 2.3 Methods 2.3.1 Enzymatic assay conditions To perform the enzymatic assay, 10 L of iNOS stock solution was added to 80 L of HEPES buffer pH = 7.4, containing 0.1 mM CaCl2 , 1 mM DTT, 0.5 mg/mL BSA, 10 M FMN, 10 M FAD, 30 M BH4 , 10 g/mL CaM, and 11 M Larginine. For the citrulline radiometric detection, 2 L of L-[H]3 arginine s.s. were added. To measure enzyme inhibition, a 10 L solution of 1400W (0.001–100 M) was added to the enzyme assay solution, followed by a pre-incubation time of 15 min at room temperature. The reaction was initiated by addition of 10 L of NADPH 7.5 mM and was carried out at 37⬚C for 20 min. Then it was stopped by following Method a) for the citrulline fluorimetric detection or Method b) for the radiometric detection. Method a): 500 L of ice-cold CH3 CN was added to the reaction and the mixture was brought to dryness under vacuum and eventually stored at –20⬚C, before the fluorescence derivatization. Method b): 500 L of ice-cold HEPES Na+ pH = 6.0 were added to the reaction, and L-[H]3 citrulline was www.jss-journal.com
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separated by DOWEX, which was washed with 1.2 mL of water. A 500 L aliquot of the eluate was added to 4 mL of scintillation fluid and the radioactivity was counted in a liquid scintillation counter. All assays were performed in triplicate. 2.3.2 Fluorescence derivatization The OPA/NAC reagent was prepared by mixing for 90 min, 5 mL of methanolic OPA solution and 20 mL of 0.2 M borate buffer containing 0.1 g of NAC. Final pH = 9.3 ± 0.05; the molar ratios of OPA to NAC was 1:3. The OPA/NAC solution was stored at 4⬚C and saved for no longer than seven days. Immediately before the HPLC analysis, the enzymatic assay residue was dissolved in 600 L of HPLC-grade water, and centrifuged at 6000 rpm for 35 min. Then 190 L of supernatant or amino acid stock solution and 60 L of OPA/NAC solution were stirred for 5 min and injected into column. 2.3.3 Apparatus and chromatographic conditions HPLC analyses were performed using a Waters (Milford, MA, USA) system composed of a P600 model pump, a 2475 multi-fluorescence detector, and a 7725i model sample injector (Rheodyne, Cotati, CA, USA) equipped with a 5 L loop. The analyses were performed on an XTerra MS C8 column (250 × 4.6 mm id, 5 m particle size; Waters), equipped with an XTerra MS C8 guard column (Waters). A column thermostat oven module Igloo-Cil (Cil Cluzeau Info Labo, France) was used. Chromatograms were recorded on a Fujitsu Siemens Esprimo computer and data were processed by the Empower Pro software (Waters). The column was eluted at a flow rate of 0.7 mL/min with linear gradients of buffers A (5% in CH3 CN in 15 mM sodium borate with 0.1% v/v trifluoroacetic acid, pH = 9.4) and B (50% in CH3 CN in 8 mM sodium borate with 0.1% v/v trifluoroacetic acid, pH = 9.4). The solvent gradient was: 0–20% B at 0–10 min, increasing B to 25% at 10–15 min, then to 40% at 15–20 min and to 70% at 20–28 min. This composition was maintained until t = 35 min, before being reduced to the initial 0% B composition. The mobile phase was prepared daily, filtered through a 0.45 m WTP membrane (Whatman, Maidstone, UK), sonicated and degassed before use. Column temperature was kept constant at 20⬚C. The injection volume was 5 L. The fluorescence intensity in the column eluate was monitored at 439 nm (emission) with excitation at 335 nm. 2.4 Method evaluation 2.4.1 Linearity, matrix effect study, LOD and LOQ Linearity was evaluated by the construction of calibration curves of solutions containing Citr in the range of 0.001– 100 M. The regression equation is y = 18303x + 403697. To study sample matrix effect, calibration curves of Citr validation standards in the concentration range of 0.001– C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
10 M were constructed. The regression equation for the obtained calibration curves is y = 17200x + 1419140. Proportional bias was evaluated as the ratio between validation and standard calibration curves slopes, while constant bias was determined as (validation curve intercept – calibration curve intercept) / validation curve slope. The LOD value was calculated from the residual SD of the regression line () of the analytical curve and its slope, according to the equations LOD = 3.3 (/S) and LOQ = 10(/S). 2.4.2 Accuracy and precision Accuracy was determined by analyzing in triplicate validation standards spiked with 0.5, 1 and 5 M Citr. Each sample was analyzed in three separate runs. Accuracy was calculated as: 100 × (overall measured concentration – unspiked native concentration)/ added concentration. The precision of the method was calculated by three validation standard solutions at 0.5, 1 and 5 M Citr. The intraday precision was calculated by six consecutive injections of each solution in one day, while the inter-day precision was evaluated by three injections of the same solution kept at 4⬚C, on three consecutive days. 2.4.3 Stability Stability was checked by injecting after one and three days 1 M Citr-derivatized sample solutions stored at 4⬚C. Peak areas were compared to those of freshly prepared samples. 2.4.4 Statistical analysis Statistical analysis of the data was achieved by using Prism 5 (GraphPad Software, La Jolla, CA, USA). The findings were regarded as significant if P values were < 0.05.
3 Results and discussion 3.1 HPLC conditions NOS catalyze the conversion of 1 equiv. of L-arginine into 1 equiv. of NO and L-citrulline, so the RPLC fluorescence detection of Citr levels represents an easy evaluation of the enzymatic activity. Our first goal was to optimize HPLC conditions to obtain a complete resolution of the OPA/NAC derivatized Citr in the enzymatic assay mixture. L-Citrulline and L-arginine standard solutions were used to set up the method, and different mobile phase compositions and ratios were attempted: phosphate and acetate buffers (from pH = 2 to 7.5) provided poor sensitivity and/or resolution, while sodium borate (pH = 9.4, 15 mM) gave the best results. The addition of 0.1% v/v of TFA ameliorated the peak shape. We adopted CH3 CN as the organic modifier, and as isocratic elution did not afford resolution of derivatized Arg and Citr, we decided to apply a mild gradient of two mobile phases A and B, www.jss-journal.com
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Table 1. Accuracy of determination of L-citrulline in NOS mixture assay (n = 9)
Citrulline added (M)
Measured (M)
Recovery (%)
0.1 1 5
0.113 1.078 4.921
97.8 106.3 98.4
Table 2. Intra- and inter-day precision of determination of L-citrulline in NOS mixture assay
Intra-day precision (n = 6)% RSD Inter-day precision (n = 9)% RSD
0.1
1
5
2.05 3.40
1.24 2.80
0.97 2.94
adopted method. The theoretical plate number for OPA/NAC derivatized amino acids was more than 5000. 3.3 Linearity, LOD and LOQ
Figure 2. Chromatograms of derivatized L-citrulline (A) and Larginine (B) standard solutions.
Figure 3. Chromatogram of a derivatized enzymatic assay mixture in absence of inhibitors (control).
containing, respectively, 5 and 50% of CH3 CN in sodium borate with 0.1% v/v of TFA.
3.2 System suitability Figure 2 shows typical chromatograms of amino acid calibration standard solutions, and Fig. 3 represents a chromatogram of an enzymatic assay mixture in absence of inhibitors (control). The retention times of derivatized Citr and Arg were 15 and 17 min, respectively, and the absence of overlapping or interfering signals supports the selectivity of the
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Linearity was evaluated for derivatized Citr standard solutions in the range of 0.001–100 M. Straight regression line with correlation coefficient of 0.999 was obtained. The Ftest applied for calibration curve and data was 2118 and provides conclusive evidence of a linear relationship between concentration and instrumental response. The LOQ was 183 nmol/L, while a value of 60 nmol/L was calculated as LOD. To evaluate bias derived from sample matrix effects, Citr spiked enzymatic assay mixtures in the range of 0.001–10 M were used to obtain calibration curves. The correlation coefficient of the obtained regression line was 0.993. Proportional bias was found, due to the different slopes in standard and validation calibration curves and it was 0.940, while constant bias was 55.480. The LOD obtained from validation standard curve was 85 nmol/L, and the LOQ was 258 nmol/L. 3.4 Accuracy and precision In order to measure accuracy and precision of the method, triplicate of 0.5, 1 and 5 M Citr validation standard solutions were analyzed. Recovery results are summarized in Table 1. Intraday and interday precision are expressed as the percentage RSD% for Citr peak area and data are reported in Table 2.
3.5 Stability Derivatized sample solutions proved to be stable for three days at least at 4⬚C. Indeed, recovery was 106.7 and 105% after one and three days, respectively.
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Figure 5. Radiometric evaluation of the dose-dependent inhibition of iNOS by 1400W. The results are from three independent experiments and are expressed as means ± standard deviations.
were stopped with cold HEPES sodium, then purified on ion exchange resins and finally added scintillation fluid. The obtained IC50 curve is shown in Fig. 5, and the extrapolated IC50 value was 0.09 M, similar to that obtained by the fluorescence detection.
4 Concluding remarks Figure 4. (A) Peak area values of derivatized L-citrulline in the presence of different 1400W concentrations in comparison with the absence of inhibitor (CTRL) and blank (BLK). The results are from three independent experiments and are expressed as means ± standard deviations. (B) Fluorimetric RPLC evaluation of dosedependent inhibition of iNOS by 1400W. The results are from three independent experiments and are expressed as means ± standard deviations.
3.6 Evaluation of 1400W IC50 To assess the suitability of our chromatographic method in the evaluation of NOS inhibitors potency, 1400W was used as a test compound. In typical enzymatic reaction conditions, different 1400W concentrations were incubated with iNOS. After incubation, reactions were stopped with ice-cold CH3 CN, dried and if necessary, stored at –20⬚C. Before the analysis, the solid residue was suspended in HPLC-grade water, derivatized with OPA/NAC reagent and analyzed. Figure 4A shows the results of the fluorescence detection, after RPLC separation, of derivatized L-citrulline as the product of iNOS activity in the absence (control) of inhibitor and in the presence of different of W1400 concentrations; and Fig. 4B shows the obtained dose-dependent inhibition of the enzyme by 1400W. An IC50 value of 0.08 M was calculated, comparable to the published data [13]. For comparison, we also evaluated IC50 by the well established radiometric assay, which is the most employed method in the evaluation of NOSs inhibitors potency. After incubation, reactions C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The selective inhibition of both neuronal and/or inducible NOS could be a new therapeutic opportunity in different diseases, so precise, reproducible, fast, and economic screening of new inhibitors is worthwhile. Here we have demonstrated a convenient method to evaluate in vitro NOS activity, alternative to the most used radiometric assay. In particular, we avoid the presence of any radioactive materials, expensive scintillation fluid and time-consuming chromatographic purification steps. The proposed derivatization step can be totally automated using an autosampler and the obtained solutions proved to be stable at 4⬚C for three days at least. So this successful RP-HPLC method with Citr fluorescence detection can be used as a reliable procedure for screening the biological activity of NOS inhibitors. This study was supported by University “G. d’Annunzio” of Chieti local grants. The authors have declared no conflict of interest.
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