- Email: [email protected]
Determination of nitrotyrosine and related compounds in biological specimens by competitive enzyme immunoassay
NITRIC OXIDE
Biology and Chemistry
Nitric Oxide 7 (2002) 11–17 www.academicpress.com
Determination of nitrotyrosine and related compounds in biological specimens by competitive enzyme immunoassay Hajime Inoue,a,* Ken-ichi Hisamatsu,b Kazumasa Ando,a Ryo Ajisaka,b and Norio Kumagaia a
Department of Plastic and Reconstructive Surgery, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae, Kawasaki 216-0015, Japan b Department of Otorhinolaryngology, Nihon University School of Medicine, 1-8-13 Surugadai, Kanda, Chiyoda, Tokyo 101-8309, Japan Received 10 April 2001; received in revised form 6 January 2002
Abstract A gas mediator, nitric oxide is converted to peroxynitrite in the presence of superoxide anion. Peroxynitrite is a potent oxidant, which injures various tissues and organs by nitration of the tyrosine residues of proteins, and it enhances the late response of inflammation. The determination of nitrated tyrosine, nitrotyrosine, which is a stable final metabolite of peroxynitrite, provides an important indicator of tissue disorders caused by peroxynitrite. This paper reports a competitive solid-phase immunoassay for measuring nitrotyrosine in various biological specimens. In this study, peroxidase-conjugated nitrotyrosine was prepared by reaction of nitrotyrosine with 1,4benzoquinone treatment, and then it was allowed to compete with nitrotyrosine on an anti-nitrotyrosine antibody-coated 96-well multiplate. No amino acids or related compounds tested in the experiments interfered with the immune reaction of nitrotyrosine, except cysteine, which only slightly inhibited the immune reaction at the concentrations higher than 1000 times the concentration of nitrotyrosine. The limit of detection of free nitrotyrosine was approximately 500 pg/mL (2 nM) at a competition ratio (B/Bo %) of 80%. The newly developed enzyme immunoassay (EIA) method was used for assay of nitrotyrosine in biological specimens, with the following results: (i) Lipopolysaccharide (LPS) activation of RAW264.7 cells induced a significant increase in nitrotyrosine production compared to that with nonactivated cells. N x -nitro-L -arginine methyl ester decreased nitrotyrosine production with either LPS-activated or nonactivated RAW cells. There is a relationship between nitrotyrosine production and nitrite ion. (ii) The nitrotyrosine level detected in the plasma specimens from healthy volunteers was 35:21 4:87 ng=mL (135:4 18:7 nM). (iii) The concentration of nitrotyrosine in the nasal lavage fluid of allergic rhinitis patients was 41:40 20:96 ng=mL (159:02 80:6 nM). Thus, the EIA method combines sensitivity and specificity with the ability to process a large number of specimens to quantify nitrotyrosine produced with in vivo and in vitro sources. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Nitrotyrosine; EIA; RAW264.7; Plasma; Nasal lavage; Nitric oxide; Peroxynitrite
A gas mediator, nitric oxide [1,2] affects not only the circulatory system [3,4] but various physiological functions and disease which include the immune system [5]. After nitric oxide is converted to nitrite ion in the presence of water, the nitrite ion is oxidized to nitrate ion because the nitric oxide content of biological specimens is usually measured in the form of nitrate and nitrite ions [6]. In the presence of superoxide anion nitric oxide is also converted to peroxynitrite [7,8]. Peroxynitrite is a potent oxidant that injures various tissues and
*
Corresponding author. Fax: +81(44)-975-3290. E-mail address: [email protected] (H. Inoue).
organs and enhances the late response of inflammation. Peroxynitrite induces nitration of the free type of tyrosine, an essential amino acid, and tyrosine residues of proteins and peptides. It is well known that the converted nitrotyrosine is a stable final metabolite of peroxynitrite. Although nitric oxide relaxes the vascular smooth muscle and the tracheal smooth muscle, nitric oxide itself does not injure any organ or cell. However, peroxynitrite, which is produced by the contact of nitric oxide and superoxide anion, severely injures various tissues and prolongs inflammation [9–12]. Accordingly, the pharmacological actions of nitric oxide should be considered in connection with the effects of peroxynitrite, as well as those of nitric oxide itself. The authors
1089-8603/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 8 9 - 8 6 0 3 ( 0 2 ) 0 0 0 0 5 - 8
12
H. Inoue et al. / Nitric Oxide 7 (2002) 11–17
have already reported that nitric oxide is involved with the occurrence of tissue disorders surrounding thermal injuries [13]. It has also been suggested that peroxynitrite and nitrotyrosine are produced around wounds of thermal injury [13]. Fortunately, nitrotyrosine is a stable marker of nitric oxide production as well, and its detection may serve as an important indicator of tissue injury. Attempts have been made to measure of nitrotyrosine by the high-performance liquid chromatography (HPLC)–amperometry method [14], which allows simultaneous detection of both amino acids, tyrosine and nitrotyrosine. The nitrotyrosine/tyrosine ratio in biological specimens, i.e., the nitration ratio, can be easily calculated using the method, but unknown substances eluted at the same retention time may interfere with the measurement of these compounds. Protein-bound nitrotyrosine cannot be measured by HPLC unless the protein is hydrolyzed, because the method is designed to detect free amino acids. Another method, immunoassay with anti-nitrotyrosine antibody, can detect both protein-bound nitrotyrosine and free nitrotyrosine. Kahn et al. [15], Steege et al. [16], and Onorato et al. [17] have reported the enzyme immunoassay (EIA)1 system for nitrotyrosine, but EIA is not considered suitable for quantitative analysis. The EIA system, however, detects the target molecule, nitrotyrosine, in both the free and protein-bound forms, making it unnecessary to hydrolyze the protein to free amino acids for the determination of nitrotyrosine. This study was designed to develop a new EIA technique for nitrotyrosine, which was used for assay of nitrotyrosine in biological specimens.
Materials and methods Reagents All reagents used were of analytical or reagent grade and were obtained from Sigma Chemical (St. Louis, MO) or Wako Pure Chemical Industries (Tokyo, Japan). Culture of RAW264.7 cells The cultured murine macrophage-like cells used, RAW264.7, were obtained from ATCC (Dainippon Pharm. Co., Osaka, Japan). Cryopreserved RAW264.7
1
Abbreviations used: NOS, nitric oxide synthase; L -NAME, N x nitro-L -arginine methyl ester; EIA, enzyme immunoassay; a-M-Tyr, amethyltyrosine; FBS, fetal bouine serum; LPS, lipopolysaccharide; ATCC, American Type Culture Collection; DME, Dulbecco’s modified Eagle’s medium; PBS, phosphate-buffered saline; ANOVA, analysis of variance; BSA, bovine serium albumin; iNOS, inducible nitric oxide synthase.
cells were thawed in water bath at 37 °C and incubated with DMEM containing 10% fetal bovine serum (FBS) in culture dishes at 37 °C under conditions of 5% carbon dioxide and 100% humidity until confluence. The old medium was replaced with fresh medium every 2 days. When the RAW264.7 cells reached confluence, they were subcultured at a six-fold dilution onto 24-well multiplates by the usual technique and used for all these experiments. Specimen preparation Blood After informed consent was obtained, heparinized blood specimens were collected from nine healthy volunteers (male/female; 5/4; age; 20–45 years). The blood was centrifuged for 10 min at 3000 rpm and 4 °C, and the separated plasma was maintained at )80 °C until use. Nasal lavage fluid After obtaining informed consent, nasal lavage was performed in 20 allergic rhinitis patients (male/female; 11/9; age: 5–67 years). The nasal cavity was washed with 5 mL of saline, and lavage fluid specimens were obtained with a suction catheter. The specimens were immediately stored at )80 °C until use. Optimization of the nitrotyrosine EIA technique Titration experiments Each well of a 96-well microtiter plate (MAXI-SORP, Nunc, Roskilde, Denmark) was coated with 0.1 mL of 1 lg=mL rabbit anti-nitrotyrosine IgG polyclonal antibody (Upstate Biol. Co., MO) diluted with 0.05 M sodium bicarbonate buffer (pH 9.6). After incubation at 4 °C overnight, the plates were rinsed once with 0.01 M phosphate-buffered saline (PBS) containing 0.05% Tween-20 (wash buffer) and then blocked with 1% bovine serum albumin in PBS at 37 °C for 1 h. The antibody-coated plates were rinsed once with wash buffer, then 0.1 mL of 100- to 102,400-fold peroxidase-conjugated nitrotyrosine was added to each well. The plates were then incubated at various times and temperatures, i.e., ambient temperature or 4 °C overnight or 37 °C for 1 h. After incubation, the plates were rinsed with wash buffer five times, and enzyme activity of the peroxidase bound to each well was determined with OPD reagents (Sumitomo Bakelight Co., Aomori, Japan). Optical density was measured at 490 nm on a micro-plate reader (Japan Bio-Rad Co., Tokyo, Japan). In these preliminary experiments, the condition obtained the highest optical density was selected. Establishment of EIA The optimal conditions of the above experiment (Titration Experiments) were incubation of 0.05 mL of
H. Inoue et al. / Nitric Oxide 7 (2002) 11–17
13
authentic nitrotyrosine at the final concentrations 1000 ng/mL–10 pg/mL and the same volume of 51,200fold peroxidase-conjugated nitrotyrosine at 4 °C overnight. The plates were allowed to immune reaction, as described under Titration experiments (above).
portion of the medium was used for the nitrite determination by the Griess method, and the remainder was used for the nitrotyrosine determination.
Preparation of peroxidase-conjugated nitrotyrosine
All data are expressed as means standard deviation (SD). The statistical significance of the results was evaluated by Dunnett’s multiple range tests after ANOVA test.
Peroxidase-conjugated nitrotyrosine was prepared according to the method of Chevrier et al. [17] by the following one-step procedure: 0.01 mL of a mixture consisting of 10 mg of ethanol solution per mL of 1,4benzoquinone was added to 0.1 mg of nitrotyrosine dissolved in 0.1 mL of 0.1 M potassium phosphate (pH 4.5). The mixture was allowed to stand at ambient temperature for 1 h in the dark, and 10 mg of horseradish peroxidase in 0.5 mL of decahydrate sodium borate potassium dihydrogenphosphate buffer (pH 9.0) was then added to the mixture. After incubation at ambient temperature overnight, the mixture was dialyzed against Hank’s balanced salt solution for removal of free nitrotyrosine. iNOS induction in RAW264.7 cells RAW264.7 cells were maintained at 37 °C under 5% carbon dioxide in the air in DMEM containing 10% FBS and seeded onto 24-well culture plates at a cell density of 2 106 cells/well. The cells were cultured for 24 h under the same conditions and then incubated with the new medium containing 10 lg=mL LPS (Escherichia coli, O111; B4) for another 24 h. After centrifugation, a
Statistical analysis
Results Detection of nitrotyrosine by EIA Optimal conditions of the nitrotyrosine determination (Figs. 1 and 2 and Table 1) The optimal incubation time and temperature were determined by using multi-EIA plates coated with 1 lg=mL of anti-nitrotyrosine antibody, and the optimal concentration of peroxidase-conjugated nitrotyrosine was also determined. As shown in Fig. 1, the best incubation time and temperature were overnight and 4 °C, respectively. The concentration of peroxidase-conjugated nitrotyrosine used was approximately 0:2 lg=mL, i.e., a 51,200-fold dilution ratio. The best sensitivity was obtained under the above-described conditions, and the procedure is shown in Table 1. As shown in Fig. 2, the limit of detection of a typical calibration curve was approximately 500 pg/mL when the S/N ratio was defined at competition ratio (B/Bo %) of 80%.
Fig. 1. Comparative study of optimum conditions for the nitrotyrosine determination. The three curves show the competition between the peroxidase-conjugated nitrotyrosine and anti-nitrotyrosine antibody immobilized onto 96-well micro plates, which are incubated at various temperatures and times. Peroxidase-conjugated nitrotyrosine was prepared by the technique described under Materials and methods. The experiments are represented as mean absorbance of four wells. The X axis represents the dilution ratio of peroxidase-conjugated nitrotyrosine. The highest S/N ratio was obtained under conditions of 18 h and 4 °C.
14
H. Inoue et al. / Nitric Oxide 7 (2002) 11–17
Nitrotyrosine production with RAW264.7 macrophage cell line (Fig. 3) Production of nitrite ion, i.e., NO metabolites, with the RAW264.7 cells cultured with LPS was increased, whereas nitrite ion production with the LPS-activated RAW cells cultured with L -NAME was decreased compared to the production in the absence of L -NAME, as shown in Fig. 3. The nitrotyrosine contents of the RAW cells cultured in the medium also showed the same pattern as that of the nitrite ion content, as shown in Fig. 3. Nitrotyrosine contents of biological specimens
Fig. 2. Typical calibration curve of nitrotyrosine. 1 lg/mL of anti-nitrotyrosine antibody was coated on a 96-well microplate. Each 0.05 mL of peroxidase-conjugated nitrotyrosine diluted 51,200-fold and 0.01– 1000 ng/mL of authentic nitrotyrosine was incubated onto the anti-nitrotyrosine antibody-coated microplate at 4 °C for 18 h. After incubation, the plate was rinsed with wash buffer five times, and enzyme activities of peroxidase bound onto each well were determined with OPD reagents. The optical density was measured at 490 nm on a microplate reader.
Table 1 Assay procedure for nitrotyrosine EIA
Cross-reactivity (Table 2) The cross-reactivity of nitrotyrosine with various amino acids and tyrosine-related compounds by the EIA method was investigated. As shown in Table 2, no amino acids or related compounds tested in the experiments interfered with the immune reaction of nitrotyrosine, except cysteine, which only slightly inhibited the immune reaction at concentrations higher than 1000 times the concentration of nitrotyrosine. As a result, there were no problems with the sensitivity or specificity of the assay.
The EIA method we developed was used for the determination of nitrotyrosine in the nasal lavage fluid of allergic rhinitis patients and in the normal human plasma. Nitrotyrosine was detected in both specimens. The nitrotyrosine concentration in the plasma of the nine healthy volunteers was 35:21 4:87 ng=mL (135:4 18:7 nM). The concentration in the nasal lavage of the 20 allergic rhinitis patients was 41:40 20:96 ng=mL (159:02 80:6 nM).
Discussion Nitration of tyrosine or tyrosine residues of proteins and peptides damages tissues and organs. Tissue nitration is mediated nonenzymatically with peroxynitrite, which is produced by reaction of nitric oxide with superoxide anion, and it impairs the normal physiological function. Since the content of nitrated compounds is known to depend on the peroxynitrite content and myeloperoxidase activity, the measurement of the nitrated compound nitrotyrosine is used as an indicator of tissue damage. Nitrotyrosine has been measured by various methods that can be roughly classified into two types. One of them is the ordinary method of common use, but it was designed to separate nitrotyrosine and other compounds by HPLC. The compounds were detected from the specific UV absorbance of amino acids [19,20], the specific fluorescence of nitrotyrosine-fluorescence derivatives [20], or the electrochemical properties of nitrotyrosine [14]. The DAB-nitrotyrosine derived from dabsyl chloride has also been used for the specific determination of nitrotyrosine [21]. These techniques allow the simultaneous assay of nitrotyrosine and tyrosine. All of the methods of detection, except UV detection, have excellent specificity, but none of them are capable of determining nitrotyrosine in proteins or peptides. To measure the nitrotyrosine in such compounds, they must be completely hydrolyzed first to their component amino acids.
H. Inoue et al. / Nitric Oxide 7 (2002) 11–17
15
Table 2 Interference of various amino acids on immune reaction of nitrotyrosine (B/Bo %)
N-Tyr Cys Arg Phe Tyr Trp His Met Val Pro a-M-Tyr
0:02 lM
0:2 lM
2:0 lM
88:7 2:7
51:32 0:1
13:03 0:7
0:1 mM
1 mM
1:0 mM
92:37 5:6 97:71 1:5 96:99 1:1 90:57 1:3 97:13 0:7 97:89 0:2 96:46 1:1 91:52 1:3 95:02 0:3 91:88 0:5
70:32 0:3 94:98 2:1 95:06 0:8 89:23 1:7 96:64 0:2 95:56 1:1 96:46 1:1 91:29 1:4 92:59 0:7 73:91 0:5
9:16 1:3 89:54 0:6 91:47 0:1 76:83 1:6 85:9 0:5 69:6 0:4 95:24 0:8 91:16 0:5 95:06 0:5 100:5 0:0
Note. a-M-Tyr, a-methyltyrosine.
Fig. 3. Relation between nitrotyrosine and nitrite contents in RAW264.7 cultured medium. RAW264.7 cells were maintained with Dulbecco’s modified Eagle’s medium containing 10% FBS under conditions of 5% carbon dioxide, 95% humidity, and 37 °C. LPS and L -NAME were added in cultured RAW cell with the fresh medium and then cultured under the same conditions for 18 h. The data obtained were expressed as the means SD of four wells. The statistical significance of the results was evaluated by Dunnett’s multiple range tests after ANOVA tests.
EIA of nitrotyrosine is excellent as a method of immunohistochemical analysis of nitrotyrosine expression and localization in tissues. It is thus considered suitable for the qualitative analysis, but not for the quantitative analysis. However, nitrotyrosine can be measured quantitatively in principle in a way similar to that for the immune reaction used in the ordinary EIA, thereby allowing EIA to be used for the determination of nitrated protein and free nitrotyrosine without hydrolysis, unlike HPLC. In the present study, the authors developed a nitrotyrosine EIA system by using a competitive binding immune reaction. Kahn et al. [15] determined nitrated protein in the plasma as biological specimens by an indirect competitive EIA for nitrotyrosine, and the new enzyme-linked immunosorbent assay technique developed by Steege
et al. [16] has been used to study the effect of diet on nitrotyrosine production in celiac diseases. Both methods are useful, but Kahn’s method [14] is premised on the synthesis of nitrotyrosine-conjugated BSA as the EIA calibration standard. Steege et al. [16] measured nitrotyrosine with an avidin–biotin system after reaction of the nitrotyrosine-related compounds with two different types of monoclonal antibodies, i.e., a solid-phased antibody and a biotinylated antibody, but the method does not allow the determination of free nitrotyrosine, because each antibody must recognize at least two different regions of free nitrotyrosine. The authors designed their new EIA system by utilizing a competitive immune reaction to overcome these shortcomings. Competitive immunoassay requires that nitrotyrosine should be labeled with some enzymes or radioisotopes, but the labeling techniques have already been established in these two cases. In the present study, the authors labeled nitrotyrosine with peroxidase, which is commonly used as a labeling enzyme for competitive EIA system of monoamine and eicosanoids [22,23]. Chevrier et al. [18] determined histamine by using peroxidase-conjugated histamine as a tracer, and Pradelles et al. [23] and Morel et al. [22] determined cholinesterase-conjugated eicosanoids and histamine, respectively. When low-molecular-weight substances are measured by EIA, either acylation or acetylation is used to increase its sensitivity and specificity [24]. However, approximately 500 pg/mL (2 pmol/mL, 2 nM) was the detection limit for free nitrotyrosine by our method without acylation. The sensitivity level obtained was (for free nitrotyrosine) approximately 100–1000 times higher than that by Kahn’s method [15], and 1/10 times (same conversion) lower than that by Steege’s technique [16]. In a brief review Herce-Pogliai et al. [25] reported only approximately 1:6–0:2 lM as the detection limit of free nitrotyrosine obtained by the HPLC-UV technique and 0.0004–50 pmol/injection by ECD, fluorescence prelabeled NBD, and GC/MS techniques. Also, this assay
16
H. Inoue et al. / Nitric Oxide 7 (2002) 11–17
system was not interfered before addition of the 10- to 100-fold concentration of physiological plasma cysteine, showing that cysteine had a problem with the plasma nitrotyrosine determination using the EIA system. These results suggest that our method has the same detection limit as that for other HPLC methods except the UV technique. In fact, Steege et al. [16] reported that no free nitrotyrosine could be detected in plasma, and Kahn et al. [15] obtained 0:12 0:01 lM in plasma by using nitrated-BSA as the calibration standard. Although Fukuyama et al. [26] were unable to detect free nitrotyrosine in plasma by the HPLC-UV technique, the HPLC-NBD method of Kamisaki et al. [20] revealed the plasma nitrotyrosine content of 31:0 6:0 pmol=mL in healthy volunteers. The plasma nitrotyrosine level obtained in healthy volunteers by the present method was 135:4 18:7 pmol=mL (0:135 0:0187 lM), being consistent with those obtained by Kamisaki et al. [20] and Kahn et al. [15]. Their results were considered almost the same as ours, although our data were slightly high. Nitrotyrosine levels in nasal lavage fluid could also be measured by our procedure, and the values obtained were higher than the plasma levels. Nitric oxide levels in the nasal lavage fluid of nasal allergy patients are known to be increased, while Sato et al. [27] detected nitrotyrosine in the nasal mucosa of allergy patients. They could not measure nitrotyrosine in the nasal lavage fluid by their low-sensitivity method. Our method needs no surgical invasion, because our method allows detection of nitrotyrosine even in the nasal lavage without collection of nasal mucosa. Under these circumstances, the measurement of nitrotyrosine in the nasal lavage may allow to elucidate the mechanism of ciliary disorders of nasal mucosa due to peroxynitrite at nasal allergic reaction. It is well known that LPS-stimulated RAW264.7 cells increase significantly nitric oxide production with inducible nitric oxide synthasse (iNOS) induction. Using this EIA technique, we investigated the influence of the activated RAW264.7 cells on nitrotyrosine production accompanying nitric oxide production. Nitric oxide and nitrotyrosine production with LPS-treated RAW264.7 cells was significantly increased after iNOS induction, but L -NAME, a NOS inhibitor, inhibited strongly nitric oxide production with RAW264.7 cells, regardless of LPS stimulation. On the other hand, nitrotyrosine production with RAW264.7 cells also changed in proportion to nitric oxide production. When nitrotyrosine was plotted along the X axis, and nitric oxide, along the Y axis in Fig. 3, NO production by RAW264.7 cells was proportional to nitrotyrosine production. The numbers of the experimental subjects should be increased for exact acquisition of the statistical correlation. Although nitrotyrosine was synthesized by either nitrite ion/tyrosine without superoxide anion or myeloperoxidase, it was confirmed that nitrotyrosine production is influenced by
nitric oxide production. The result suggests that the method is a useful procedure for elucidating the mechanism of tissue disorders due to peroxynitrite. The mechanism of tissue disorders accompanying peroxynitrite production in various diseases is under investigation by using this method. Furthermore, calculation of the nitration ratio (nitrotyrosine/1000 tyrosine residues) is very important as an indicator of tissue damage. A discriminative EIA method that allows the simultaneous assay of tyrosine and nitrotyrosine is under development.
References [1] R.F. Furchgott, J.V. Zawadzki, The obligatory role of endothelial cells in the relaxation of arterial muscle by acetylcholine, Nature 228 (1980) 373–377. [2] R.M.J. Palmaer, A.G. Ferrig, S. Moncada, Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor, Nature 327 (1987) 524–526. [3] P. Vallance, J. Collier, S. Moncada, Effects of endotheliumderived nitric oxide on peripheral arteriolar tone in man, Lancet II (1989) 997–1000. [4] D.C. Lefroy, Craket, N.G. Uren, Effects of inhibition of nitric oxide synthesis on epicardial coronary artery caliber and coronary blood flow in humans, Circulation 8 (1993) 43–54. [5] H. Kolb, V. Kolb-Bachofen, Nitric oxide: a pathogenic factor in autoimmunity, Immunol. Today 13 (1992) 157–160. [6] L.C. Green, D.A. Wagner, J. Glogowski, P.L. Skipper, J.S. Wishnok, S.R. Tannernbaum, Analysis of nitrate, nitrite, and 15N-nitrate in biological fluids, Anal. Biochem. 126 (1992) 131– 138. [7] H. Ischiropoulos, L. Zhu, J.S. Beckman, Peroxynitrite formation from macrophage-derived nitric oxide, Arch. Biochem. Biophys. 298 (1992) 446–451. [8] H. Ischiropoulos, Zhu, J. Chen, M. Tsai, J.C. Martin, C.D. Smith, J.S. Beckman, Peroxy-nitrite-mediated tyrosine nitration catalyzed by superoxide dismutase, Arch. Biochem. Biophys. 298 (1992) 431–437. [9] R. Radi, J.S. Beckman, K.M. Bush, B.A. Freeman, Peroxy nitric oxidation of sulfohydryls. The cytotoxic potential of superoxide and nitric oxide, J. Biol. Chem. 266 (1991) 4244–4250. [10] H. Ohshima, M. Friesen, I. Brouet, H. Bartsh, Nitrotyrosine as anew marker for endogenous nitrosation and nitration of proteins, Fd. Chem. Toxic. 28 (1990) 647–652. [11] N.W. Kooy, J.A. Royall, Y.Z. Ye, D.R. Kelly, J.S. Beckman, Evidence for in vivo peroxynitirite production in human acute injury, Am. J. Respir. Crit. Care Med. 151 (1995) 1250–1254. [12] H. Kuar, B. Halliwell, Nitrotyrosine in serum and synovial fluid from rheumatoid patients, FEBS Lett. 350 (1994) 9–12. [13] H. Inoue, K. Ando, N. Wakisaka, K. Matsuzaki, M. Aihara, N. Kumagai, Effects of nitric oxide synthase inhibitors on vascular hyperpermeability with thermal injury in mice, Nitric Oxide 5 (2001) 334–342. [14] M.K. Shigenaga, H.H. Lee, B.C. Blount, S. Christen, E. Shigeno, H. Yip, B.N. Ames, Inflammation and NOx-induced nitration: Assay for3-nitrotyrosine by HPLC with electrochemical detection, Proc. Natl. Acad. Sci. USA 94 (1997) 3211–3216. [15] J. Kahn, D.M. Brennan, N. Bradley, B. Gao, R. Bruckdorfer, M. Jacobs, 3-nitrotyrosine in the proteins of human plasma determined by an ELISA method, Biochem. J. 330 (1998) 795– 801. [16] J.C.A. Ter Steege, L. Koster-Kamphus, E.A. van Straaten, P.P. Forget, W. Biirman, Nitrotyrosine in plasma of celiac disease
H. Inoue et al. / Nitric Oxide 7 (2002) 11–17
[17]
[18]
[19]
[20]
[21]
patients as detected by a new sandwich ELISA, Free Radical Biol. Med. 25 (1998) 953–963. J.M. Onorato, S. Thorpe, J.W. Baynes, Immunohistochemical and ELISA assays for biomarkers of oxidative stress in aging and disease, Ann. NY. Acad. Sci. 854 (1998) 277–290. D. Chevrier, J.L. Guesdon, J.C. Mazie, S. Avrameas, Enzyme immunoassay for the measurement of histamine, J. Immnol. Methods 94 (1986) 119–125. N. Fukuyama, K. Ichimori, Z. Su, H. Ishida, H. Nakazawa, Peroxynitrite formation from activated human leukocytes, Biochem. Biophys. Res. Commun. 224 (1996) 414–419. Y. Kamisaki, K. Wada, K. Nakamoto, Y. Kishimoto, M. Kitano, T. Itoh, Sensitive determination of nitrotyrosine in human plasma by isocratic high-performance liquid chromatography, J. Chromatogr. B 685 (1996) 343–347. H. Inoue, O. Kunihiro, M. Tomioka, N. Kumagai, Rapid and selective simultaneous determination of nitrotyrosine and tyrosine by a dabsylation technique, Jpn. J. Pharmacol. 82 (suppl. 1) (2000) 150.
17
[22] A. Morel, M. Darmon, M. Delaage, An immunoenzymoassay for histamine, Agents Action 30 (1990) 291–293. [23] P. Pradelles, J. Grassi, J. Maclouf, Enzyme immunoassays of eicosanoids using acetylcholine esterase as label: an alternative to radioimmunoassay, Anal. Chem. 57 (1985) 1170– 1173. [24] P. Pradelles, J. Grassi, Enzyme immunoassays of adenosine cyclic 30 50 monophosphate using acetylcholinesterase, Anal. Chem. 61 (1989) 447. [25] C. Herce-Pagliai, S. Kotecha, D.E.G. Shuker, Analytical methods for 3-nitrotyrosine as a marker of exposure to reactive nitrogen species: a review, Nitric Oxide 2 (1998) 324–336. [26] N. Fukuyama, Y. Teakebayashi, M. Hida, H. Ishida, K. Ichimori, H. Nakazawa, Clinical evidence of peroxynitrite formation in chronic renal failure patients with septic shock, Free Radical Biol. Med. 22 (1997) 771–774. [27] M. Sato, N. Fukuyama, M. Sakai, H. Nakazawa, Increased nitric oxide in nasal mucosa-indices for severe perennial nasal allergy, Clin. Exp. Allerg. 28 (1998) 597–605.
Biology and Chemistry
Nitric Oxide 7 (2002) 11–17 www.academicpress.com
Determination of nitrotyrosine and related compounds in biological specimens by competitive enzyme immunoassay Hajime Inoue,a,* Ken-ichi Hisamatsu,b Kazumasa Ando,a Ryo Ajisaka,b and Norio Kumagaia a
Department of Plastic and Reconstructive Surgery, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae, Kawasaki 216-0015, Japan b Department of Otorhinolaryngology, Nihon University School of Medicine, 1-8-13 Surugadai, Kanda, Chiyoda, Tokyo 101-8309, Japan Received 10 April 2001; received in revised form 6 January 2002
Abstract A gas mediator, nitric oxide is converted to peroxynitrite in the presence of superoxide anion. Peroxynitrite is a potent oxidant, which injures various tissues and organs by nitration of the tyrosine residues of proteins, and it enhances the late response of inflammation. The determination of nitrated tyrosine, nitrotyrosine, which is a stable final metabolite of peroxynitrite, provides an important indicator of tissue disorders caused by peroxynitrite. This paper reports a competitive solid-phase immunoassay for measuring nitrotyrosine in various biological specimens. In this study, peroxidase-conjugated nitrotyrosine was prepared by reaction of nitrotyrosine with 1,4benzoquinone treatment, and then it was allowed to compete with nitrotyrosine on an anti-nitrotyrosine antibody-coated 96-well multiplate. No amino acids or related compounds tested in the experiments interfered with the immune reaction of nitrotyrosine, except cysteine, which only slightly inhibited the immune reaction at the concentrations higher than 1000 times the concentration of nitrotyrosine. The limit of detection of free nitrotyrosine was approximately 500 pg/mL (2 nM) at a competition ratio (B/Bo %) of 80%. The newly developed enzyme immunoassay (EIA) method was used for assay of nitrotyrosine in biological specimens, with the following results: (i) Lipopolysaccharide (LPS) activation of RAW264.7 cells induced a significant increase in nitrotyrosine production compared to that with nonactivated cells. N x -nitro-L -arginine methyl ester decreased nitrotyrosine production with either LPS-activated or nonactivated RAW cells. There is a relationship between nitrotyrosine production and nitrite ion. (ii) The nitrotyrosine level detected in the plasma specimens from healthy volunteers was 35:21 4:87 ng=mL (135:4 18:7 nM). (iii) The concentration of nitrotyrosine in the nasal lavage fluid of allergic rhinitis patients was 41:40 20:96 ng=mL (159:02 80:6 nM). Thus, the EIA method combines sensitivity and specificity with the ability to process a large number of specimens to quantify nitrotyrosine produced with in vivo and in vitro sources. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Nitrotyrosine; EIA; RAW264.7; Plasma; Nasal lavage; Nitric oxide; Peroxynitrite
A gas mediator, nitric oxide [1,2] affects not only the circulatory system [3,4] but various physiological functions and disease which include the immune system [5]. After nitric oxide is converted to nitrite ion in the presence of water, the nitrite ion is oxidized to nitrate ion because the nitric oxide content of biological specimens is usually measured in the form of nitrate and nitrite ions [6]. In the presence of superoxide anion nitric oxide is also converted to peroxynitrite [7,8]. Peroxynitrite is a potent oxidant that injures various tissues and
*
Corresponding author. Fax: +81(44)-975-3290. E-mail address: [email protected] (H. Inoue).
organs and enhances the late response of inflammation. Peroxynitrite induces nitration of the free type of tyrosine, an essential amino acid, and tyrosine residues of proteins and peptides. It is well known that the converted nitrotyrosine is a stable final metabolite of peroxynitrite. Although nitric oxide relaxes the vascular smooth muscle and the tracheal smooth muscle, nitric oxide itself does not injure any organ or cell. However, peroxynitrite, which is produced by the contact of nitric oxide and superoxide anion, severely injures various tissues and prolongs inflammation [9–12]. Accordingly, the pharmacological actions of nitric oxide should be considered in connection with the effects of peroxynitrite, as well as those of nitric oxide itself. The authors
1089-8603/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 8 9 - 8 6 0 3 ( 0 2 ) 0 0 0 0 5 - 8
12
H. Inoue et al. / Nitric Oxide 7 (2002) 11–17
have already reported that nitric oxide is involved with the occurrence of tissue disorders surrounding thermal injuries [13]. It has also been suggested that peroxynitrite and nitrotyrosine are produced around wounds of thermal injury [13]. Fortunately, nitrotyrosine is a stable marker of nitric oxide production as well, and its detection may serve as an important indicator of tissue injury. Attempts have been made to measure of nitrotyrosine by the high-performance liquid chromatography (HPLC)–amperometry method [14], which allows simultaneous detection of both amino acids, tyrosine and nitrotyrosine. The nitrotyrosine/tyrosine ratio in biological specimens, i.e., the nitration ratio, can be easily calculated using the method, but unknown substances eluted at the same retention time may interfere with the measurement of these compounds. Protein-bound nitrotyrosine cannot be measured by HPLC unless the protein is hydrolyzed, because the method is designed to detect free amino acids. Another method, immunoassay with anti-nitrotyrosine antibody, can detect both protein-bound nitrotyrosine and free nitrotyrosine. Kahn et al. [15], Steege et al. [16], and Onorato et al. [17] have reported the enzyme immunoassay (EIA)1 system for nitrotyrosine, but EIA is not considered suitable for quantitative analysis. The EIA system, however, detects the target molecule, nitrotyrosine, in both the free and protein-bound forms, making it unnecessary to hydrolyze the protein to free amino acids for the determination of nitrotyrosine. This study was designed to develop a new EIA technique for nitrotyrosine, which was used for assay of nitrotyrosine in biological specimens.
Materials and methods Reagents All reagents used were of analytical or reagent grade and were obtained from Sigma Chemical (St. Louis, MO) or Wako Pure Chemical Industries (Tokyo, Japan). Culture of RAW264.7 cells The cultured murine macrophage-like cells used, RAW264.7, were obtained from ATCC (Dainippon Pharm. Co., Osaka, Japan). Cryopreserved RAW264.7
1
Abbreviations used: NOS, nitric oxide synthase; L -NAME, N x nitro-L -arginine methyl ester; EIA, enzyme immunoassay; a-M-Tyr, amethyltyrosine; FBS, fetal bouine serum; LPS, lipopolysaccharide; ATCC, American Type Culture Collection; DME, Dulbecco’s modified Eagle’s medium; PBS, phosphate-buffered saline; ANOVA, analysis of variance; BSA, bovine serium albumin; iNOS, inducible nitric oxide synthase.
cells were thawed in water bath at 37 °C and incubated with DMEM containing 10% fetal bovine serum (FBS) in culture dishes at 37 °C under conditions of 5% carbon dioxide and 100% humidity until confluence. The old medium was replaced with fresh medium every 2 days. When the RAW264.7 cells reached confluence, they were subcultured at a six-fold dilution onto 24-well multiplates by the usual technique and used for all these experiments. Specimen preparation Blood After informed consent was obtained, heparinized blood specimens were collected from nine healthy volunteers (male/female; 5/4; age; 20–45 years). The blood was centrifuged for 10 min at 3000 rpm and 4 °C, and the separated plasma was maintained at )80 °C until use. Nasal lavage fluid After obtaining informed consent, nasal lavage was performed in 20 allergic rhinitis patients (male/female; 11/9; age: 5–67 years). The nasal cavity was washed with 5 mL of saline, and lavage fluid specimens were obtained with a suction catheter. The specimens were immediately stored at )80 °C until use. Optimization of the nitrotyrosine EIA technique Titration experiments Each well of a 96-well microtiter plate (MAXI-SORP, Nunc, Roskilde, Denmark) was coated with 0.1 mL of 1 lg=mL rabbit anti-nitrotyrosine IgG polyclonal antibody (Upstate Biol. Co., MO) diluted with 0.05 M sodium bicarbonate buffer (pH 9.6). After incubation at 4 °C overnight, the plates were rinsed once with 0.01 M phosphate-buffered saline (PBS) containing 0.05% Tween-20 (wash buffer) and then blocked with 1% bovine serum albumin in PBS at 37 °C for 1 h. The antibody-coated plates were rinsed once with wash buffer, then 0.1 mL of 100- to 102,400-fold peroxidase-conjugated nitrotyrosine was added to each well. The plates were then incubated at various times and temperatures, i.e., ambient temperature or 4 °C overnight or 37 °C for 1 h. After incubation, the plates were rinsed with wash buffer five times, and enzyme activity of the peroxidase bound to each well was determined with OPD reagents (Sumitomo Bakelight Co., Aomori, Japan). Optical density was measured at 490 nm on a micro-plate reader (Japan Bio-Rad Co., Tokyo, Japan). In these preliminary experiments, the condition obtained the highest optical density was selected. Establishment of EIA The optimal conditions of the above experiment (Titration Experiments) were incubation of 0.05 mL of
H. Inoue et al. / Nitric Oxide 7 (2002) 11–17
13
authentic nitrotyrosine at the final concentrations 1000 ng/mL–10 pg/mL and the same volume of 51,200fold peroxidase-conjugated nitrotyrosine at 4 °C overnight. The plates were allowed to immune reaction, as described under Titration experiments (above).
portion of the medium was used for the nitrite determination by the Griess method, and the remainder was used for the nitrotyrosine determination.
Preparation of peroxidase-conjugated nitrotyrosine
All data are expressed as means standard deviation (SD). The statistical significance of the results was evaluated by Dunnett’s multiple range tests after ANOVA test.
Peroxidase-conjugated nitrotyrosine was prepared according to the method of Chevrier et al. [17] by the following one-step procedure: 0.01 mL of a mixture consisting of 10 mg of ethanol solution per mL of 1,4benzoquinone was added to 0.1 mg of nitrotyrosine dissolved in 0.1 mL of 0.1 M potassium phosphate (pH 4.5). The mixture was allowed to stand at ambient temperature for 1 h in the dark, and 10 mg of horseradish peroxidase in 0.5 mL of decahydrate sodium borate potassium dihydrogenphosphate buffer (pH 9.0) was then added to the mixture. After incubation at ambient temperature overnight, the mixture was dialyzed against Hank’s balanced salt solution for removal of free nitrotyrosine. iNOS induction in RAW264.7 cells RAW264.7 cells were maintained at 37 °C under 5% carbon dioxide in the air in DMEM containing 10% FBS and seeded onto 24-well culture plates at a cell density of 2 106 cells/well. The cells were cultured for 24 h under the same conditions and then incubated with the new medium containing 10 lg=mL LPS (Escherichia coli, O111; B4) for another 24 h. After centrifugation, a
Statistical analysis
Results Detection of nitrotyrosine by EIA Optimal conditions of the nitrotyrosine determination (Figs. 1 and 2 and Table 1) The optimal incubation time and temperature were determined by using multi-EIA plates coated with 1 lg=mL of anti-nitrotyrosine antibody, and the optimal concentration of peroxidase-conjugated nitrotyrosine was also determined. As shown in Fig. 1, the best incubation time and temperature were overnight and 4 °C, respectively. The concentration of peroxidase-conjugated nitrotyrosine used was approximately 0:2 lg=mL, i.e., a 51,200-fold dilution ratio. The best sensitivity was obtained under the above-described conditions, and the procedure is shown in Table 1. As shown in Fig. 2, the limit of detection of a typical calibration curve was approximately 500 pg/mL when the S/N ratio was defined at competition ratio (B/Bo %) of 80%.
Fig. 1. Comparative study of optimum conditions for the nitrotyrosine determination. The three curves show the competition between the peroxidase-conjugated nitrotyrosine and anti-nitrotyrosine antibody immobilized onto 96-well micro plates, which are incubated at various temperatures and times. Peroxidase-conjugated nitrotyrosine was prepared by the technique described under Materials and methods. The experiments are represented as mean absorbance of four wells. The X axis represents the dilution ratio of peroxidase-conjugated nitrotyrosine. The highest S/N ratio was obtained under conditions of 18 h and 4 °C.
14
H. Inoue et al. / Nitric Oxide 7 (2002) 11–17
Nitrotyrosine production with RAW264.7 macrophage cell line (Fig. 3) Production of nitrite ion, i.e., NO metabolites, with the RAW264.7 cells cultured with LPS was increased, whereas nitrite ion production with the LPS-activated RAW cells cultured with L -NAME was decreased compared to the production in the absence of L -NAME, as shown in Fig. 3. The nitrotyrosine contents of the RAW cells cultured in the medium also showed the same pattern as that of the nitrite ion content, as shown in Fig. 3. Nitrotyrosine contents of biological specimens
Fig. 2. Typical calibration curve of nitrotyrosine. 1 lg/mL of anti-nitrotyrosine antibody was coated on a 96-well microplate. Each 0.05 mL of peroxidase-conjugated nitrotyrosine diluted 51,200-fold and 0.01– 1000 ng/mL of authentic nitrotyrosine was incubated onto the anti-nitrotyrosine antibody-coated microplate at 4 °C for 18 h. After incubation, the plate was rinsed with wash buffer five times, and enzyme activities of peroxidase bound onto each well were determined with OPD reagents. The optical density was measured at 490 nm on a microplate reader.
Table 1 Assay procedure for nitrotyrosine EIA
Cross-reactivity (Table 2) The cross-reactivity of nitrotyrosine with various amino acids and tyrosine-related compounds by the EIA method was investigated. As shown in Table 2, no amino acids or related compounds tested in the experiments interfered with the immune reaction of nitrotyrosine, except cysteine, which only slightly inhibited the immune reaction at concentrations higher than 1000 times the concentration of nitrotyrosine. As a result, there were no problems with the sensitivity or specificity of the assay.
The EIA method we developed was used for the determination of nitrotyrosine in the nasal lavage fluid of allergic rhinitis patients and in the normal human plasma. Nitrotyrosine was detected in both specimens. The nitrotyrosine concentration in the plasma of the nine healthy volunteers was 35:21 4:87 ng=mL (135:4 18:7 nM). The concentration in the nasal lavage of the 20 allergic rhinitis patients was 41:40 20:96 ng=mL (159:02 80:6 nM).
Discussion Nitration of tyrosine or tyrosine residues of proteins and peptides damages tissues and organs. Tissue nitration is mediated nonenzymatically with peroxynitrite, which is produced by reaction of nitric oxide with superoxide anion, and it impairs the normal physiological function. Since the content of nitrated compounds is known to depend on the peroxynitrite content and myeloperoxidase activity, the measurement of the nitrated compound nitrotyrosine is used as an indicator of tissue damage. Nitrotyrosine has been measured by various methods that can be roughly classified into two types. One of them is the ordinary method of common use, but it was designed to separate nitrotyrosine and other compounds by HPLC. The compounds were detected from the specific UV absorbance of amino acids [19,20], the specific fluorescence of nitrotyrosine-fluorescence derivatives [20], or the electrochemical properties of nitrotyrosine [14]. The DAB-nitrotyrosine derived from dabsyl chloride has also been used for the specific determination of nitrotyrosine [21]. These techniques allow the simultaneous assay of nitrotyrosine and tyrosine. All of the methods of detection, except UV detection, have excellent specificity, but none of them are capable of determining nitrotyrosine in proteins or peptides. To measure the nitrotyrosine in such compounds, they must be completely hydrolyzed first to their component amino acids.
H. Inoue et al. / Nitric Oxide 7 (2002) 11–17
15
Table 2 Interference of various amino acids on immune reaction of nitrotyrosine (B/Bo %)
N-Tyr Cys Arg Phe Tyr Trp His Met Val Pro a-M-Tyr
0:02 lM
0:2 lM
2:0 lM
88:7 2:7
51:32 0:1
13:03 0:7
0:1 mM
1 mM
1:0 mM
92:37 5:6 97:71 1:5 96:99 1:1 90:57 1:3 97:13 0:7 97:89 0:2 96:46 1:1 91:52 1:3 95:02 0:3 91:88 0:5
70:32 0:3 94:98 2:1 95:06 0:8 89:23 1:7 96:64 0:2 95:56 1:1 96:46 1:1 91:29 1:4 92:59 0:7 73:91 0:5
9:16 1:3 89:54 0:6 91:47 0:1 76:83 1:6 85:9 0:5 69:6 0:4 95:24 0:8 91:16 0:5 95:06 0:5 100:5 0:0
Note. a-M-Tyr, a-methyltyrosine.
Fig. 3. Relation between nitrotyrosine and nitrite contents in RAW264.7 cultured medium. RAW264.7 cells were maintained with Dulbecco’s modified Eagle’s medium containing 10% FBS under conditions of 5% carbon dioxide, 95% humidity, and 37 °C. LPS and L -NAME were added in cultured RAW cell with the fresh medium and then cultured under the same conditions for 18 h. The data obtained were expressed as the means SD of four wells. The statistical significance of the results was evaluated by Dunnett’s multiple range tests after ANOVA tests.
EIA of nitrotyrosine is excellent as a method of immunohistochemical analysis of nitrotyrosine expression and localization in tissues. It is thus considered suitable for the qualitative analysis, but not for the quantitative analysis. However, nitrotyrosine can be measured quantitatively in principle in a way similar to that for the immune reaction used in the ordinary EIA, thereby allowing EIA to be used for the determination of nitrated protein and free nitrotyrosine without hydrolysis, unlike HPLC. In the present study, the authors developed a nitrotyrosine EIA system by using a competitive binding immune reaction. Kahn et al. [15] determined nitrated protein in the plasma as biological specimens by an indirect competitive EIA for nitrotyrosine, and the new enzyme-linked immunosorbent assay technique developed by Steege
et al. [16] has been used to study the effect of diet on nitrotyrosine production in celiac diseases. Both methods are useful, but Kahn’s method [14] is premised on the synthesis of nitrotyrosine-conjugated BSA as the EIA calibration standard. Steege et al. [16] measured nitrotyrosine with an avidin–biotin system after reaction of the nitrotyrosine-related compounds with two different types of monoclonal antibodies, i.e., a solid-phased antibody and a biotinylated antibody, but the method does not allow the determination of free nitrotyrosine, because each antibody must recognize at least two different regions of free nitrotyrosine. The authors designed their new EIA system by utilizing a competitive immune reaction to overcome these shortcomings. Competitive immunoassay requires that nitrotyrosine should be labeled with some enzymes or radioisotopes, but the labeling techniques have already been established in these two cases. In the present study, the authors labeled nitrotyrosine with peroxidase, which is commonly used as a labeling enzyme for competitive EIA system of monoamine and eicosanoids [22,23]. Chevrier et al. [18] determined histamine by using peroxidase-conjugated histamine as a tracer, and Pradelles et al. [23] and Morel et al. [22] determined cholinesterase-conjugated eicosanoids and histamine, respectively. When low-molecular-weight substances are measured by EIA, either acylation or acetylation is used to increase its sensitivity and specificity [24]. However, approximately 500 pg/mL (2 pmol/mL, 2 nM) was the detection limit for free nitrotyrosine by our method without acylation. The sensitivity level obtained was (for free nitrotyrosine) approximately 100–1000 times higher than that by Kahn’s method [15], and 1/10 times (same conversion) lower than that by Steege’s technique [16]. In a brief review Herce-Pogliai et al. [25] reported only approximately 1:6–0:2 lM as the detection limit of free nitrotyrosine obtained by the HPLC-UV technique and 0.0004–50 pmol/injection by ECD, fluorescence prelabeled NBD, and GC/MS techniques. Also, this assay
16
H. Inoue et al. / Nitric Oxide 7 (2002) 11–17
system was not interfered before addition of the 10- to 100-fold concentration of physiological plasma cysteine, showing that cysteine had a problem with the plasma nitrotyrosine determination using the EIA system. These results suggest that our method has the same detection limit as that for other HPLC methods except the UV technique. In fact, Steege et al. [16] reported that no free nitrotyrosine could be detected in plasma, and Kahn et al. [15] obtained 0:12 0:01 lM in plasma by using nitrated-BSA as the calibration standard. Although Fukuyama et al. [26] were unable to detect free nitrotyrosine in plasma by the HPLC-UV technique, the HPLC-NBD method of Kamisaki et al. [20] revealed the plasma nitrotyrosine content of 31:0 6:0 pmol=mL in healthy volunteers. The plasma nitrotyrosine level obtained in healthy volunteers by the present method was 135:4 18:7 pmol=mL (0:135 0:0187 lM), being consistent with those obtained by Kamisaki et al. [20] and Kahn et al. [15]. Their results were considered almost the same as ours, although our data were slightly high. Nitrotyrosine levels in nasal lavage fluid could also be measured by our procedure, and the values obtained were higher than the plasma levels. Nitric oxide levels in the nasal lavage fluid of nasal allergy patients are known to be increased, while Sato et al. [27] detected nitrotyrosine in the nasal mucosa of allergy patients. They could not measure nitrotyrosine in the nasal lavage fluid by their low-sensitivity method. Our method needs no surgical invasion, because our method allows detection of nitrotyrosine even in the nasal lavage without collection of nasal mucosa. Under these circumstances, the measurement of nitrotyrosine in the nasal lavage may allow to elucidate the mechanism of ciliary disorders of nasal mucosa due to peroxynitrite at nasal allergic reaction. It is well known that LPS-stimulated RAW264.7 cells increase significantly nitric oxide production with inducible nitric oxide synthasse (iNOS) induction. Using this EIA technique, we investigated the influence of the activated RAW264.7 cells on nitrotyrosine production accompanying nitric oxide production. Nitric oxide and nitrotyrosine production with LPS-treated RAW264.7 cells was significantly increased after iNOS induction, but L -NAME, a NOS inhibitor, inhibited strongly nitric oxide production with RAW264.7 cells, regardless of LPS stimulation. On the other hand, nitrotyrosine production with RAW264.7 cells also changed in proportion to nitric oxide production. When nitrotyrosine was plotted along the X axis, and nitric oxide, along the Y axis in Fig. 3, NO production by RAW264.7 cells was proportional to nitrotyrosine production. The numbers of the experimental subjects should be increased for exact acquisition of the statistical correlation. Although nitrotyrosine was synthesized by either nitrite ion/tyrosine without superoxide anion or myeloperoxidase, it was confirmed that nitrotyrosine production is influenced by
nitric oxide production. The result suggests that the method is a useful procedure for elucidating the mechanism of tissue disorders due to peroxynitrite. The mechanism of tissue disorders accompanying peroxynitrite production in various diseases is under investigation by using this method. Furthermore, calculation of the nitration ratio (nitrotyrosine/1000 tyrosine residues) is very important as an indicator of tissue damage. A discriminative EIA method that allows the simultaneous assay of tyrosine and nitrotyrosine is under development.
References [1] R.F. Furchgott, J.V. Zawadzki, The obligatory role of endothelial cells in the relaxation of arterial muscle by acetylcholine, Nature 228 (1980) 373–377. [2] R.M.J. Palmaer, A.G. Ferrig, S. Moncada, Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor, Nature 327 (1987) 524–526. [3] P. Vallance, J. Collier, S. Moncada, Effects of endotheliumderived nitric oxide on peripheral arteriolar tone in man, Lancet II (1989) 997–1000. [4] D.C. Lefroy, Craket, N.G. Uren, Effects of inhibition of nitric oxide synthesis on epicardial coronary artery caliber and coronary blood flow in humans, Circulation 8 (1993) 43–54. [5] H. Kolb, V. Kolb-Bachofen, Nitric oxide: a pathogenic factor in autoimmunity, Immunol. Today 13 (1992) 157–160. [6] L.C. Green, D.A. Wagner, J. Glogowski, P.L. Skipper, J.S. Wishnok, S.R. Tannernbaum, Analysis of nitrate, nitrite, and 15N-nitrate in biological fluids, Anal. Biochem. 126 (1992) 131– 138. [7] H. Ischiropoulos, L. Zhu, J.S. Beckman, Peroxynitrite formation from macrophage-derived nitric oxide, Arch. Biochem. Biophys. 298 (1992) 446–451. [8] H. Ischiropoulos, Zhu, J. Chen, M. Tsai, J.C. Martin, C.D. Smith, J.S. Beckman, Peroxy-nitrite-mediated tyrosine nitration catalyzed by superoxide dismutase, Arch. Biochem. Biophys. 298 (1992) 431–437. [9] R. Radi, J.S. Beckman, K.M. Bush, B.A. Freeman, Peroxy nitric oxidation of sulfohydryls. The cytotoxic potential of superoxide and nitric oxide, J. Biol. Chem. 266 (1991) 4244–4250. [10] H. Ohshima, M. Friesen, I. Brouet, H. Bartsh, Nitrotyrosine as anew marker for endogenous nitrosation and nitration of proteins, Fd. Chem. Toxic. 28 (1990) 647–652. [11] N.W. Kooy, J.A. Royall, Y.Z. Ye, D.R. Kelly, J.S. Beckman, Evidence for in vivo peroxynitirite production in human acute injury, Am. J. Respir. Crit. Care Med. 151 (1995) 1250–1254. [12] H. Kuar, B. Halliwell, Nitrotyrosine in serum and synovial fluid from rheumatoid patients, FEBS Lett. 350 (1994) 9–12. [13] H. Inoue, K. Ando, N. Wakisaka, K. Matsuzaki, M. Aihara, N. Kumagai, Effects of nitric oxide synthase inhibitors on vascular hyperpermeability with thermal injury in mice, Nitric Oxide 5 (2001) 334–342. [14] M.K. Shigenaga, H.H. Lee, B.C. Blount, S. Christen, E. Shigeno, H. Yip, B.N. Ames, Inflammation and NOx-induced nitration: Assay for3-nitrotyrosine by HPLC with electrochemical detection, Proc. Natl. Acad. Sci. USA 94 (1997) 3211–3216. [15] J. Kahn, D.M. Brennan, N. Bradley, B. Gao, R. Bruckdorfer, M. Jacobs, 3-nitrotyrosine in the proteins of human plasma determined by an ELISA method, Biochem. J. 330 (1998) 795– 801. [16] J.C.A. Ter Steege, L. Koster-Kamphus, E.A. van Straaten, P.P. Forget, W. Biirman, Nitrotyrosine in plasma of celiac disease
H. Inoue et al. / Nitric Oxide 7 (2002) 11–17
[17]
[18]
[19]
[20]
[21]
patients as detected by a new sandwich ELISA, Free Radical Biol. Med. 25 (1998) 953–963. J.M. Onorato, S. Thorpe, J.W. Baynes, Immunohistochemical and ELISA assays for biomarkers of oxidative stress in aging and disease, Ann. NY. Acad. Sci. 854 (1998) 277–290. D. Chevrier, J.L. Guesdon, J.C. Mazie, S. Avrameas, Enzyme immunoassay for the measurement of histamine, J. Immnol. Methods 94 (1986) 119–125. N. Fukuyama, K. Ichimori, Z. Su, H. Ishida, H. Nakazawa, Peroxynitrite formation from activated human leukocytes, Biochem. Biophys. Res. Commun. 224 (1996) 414–419. Y. Kamisaki, K. Wada, K. Nakamoto, Y. Kishimoto, M. Kitano, T. Itoh, Sensitive determination of nitrotyrosine in human plasma by isocratic high-performance liquid chromatography, J. Chromatogr. B 685 (1996) 343–347. H. Inoue, O. Kunihiro, M. Tomioka, N. Kumagai, Rapid and selective simultaneous determination of nitrotyrosine and tyrosine by a dabsylation technique, Jpn. J. Pharmacol. 82 (suppl. 1) (2000) 150.
17
[22] A. Morel, M. Darmon, M. Delaage, An immunoenzymoassay for histamine, Agents Action 30 (1990) 291–293. [23] P. Pradelles, J. Grassi, J. Maclouf, Enzyme immunoassays of eicosanoids using acetylcholine esterase as label: an alternative to radioimmunoassay, Anal. Chem. 57 (1985) 1170– 1173. [24] P. Pradelles, J. Grassi, Enzyme immunoassays of adenosine cyclic 30 50 monophosphate using acetylcholinesterase, Anal. Chem. 61 (1989) 447. [25] C. Herce-Pagliai, S. Kotecha, D.E.G. Shuker, Analytical methods for 3-nitrotyrosine as a marker of exposure to reactive nitrogen species: a review, Nitric Oxide 2 (1998) 324–336. [26] N. Fukuyama, Y. Teakebayashi, M. Hida, H. Ishida, K. Ichimori, H. Nakazawa, Clinical evidence of peroxynitrite formation in chronic renal failure patients with septic shock, Free Radical Biol. Med. 22 (1997) 771–774. [27] M. Sato, N. Fukuyama, M. Sakai, H. Nakazawa, Increased nitric oxide in nasal mucosa-indices for severe perennial nasal allergy, Clin. Exp. Allerg. 28 (1998) 597–605.