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Isoprostane as a promising biomarker in pulmonary arterial hypertension: Preanalytical and analytical viewpoints. Response to letter to the editor
International Journal of Cardiology 177 (2014) 632–633
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Letter to the Editor
Isoprostane as a promising biomarker in pulmonary arterial hypertension: Preanalytical and analytical viewpoints. Response to letter to the editor Rui Zhang a, Zhi-Cheng Jing b,⁎ a b
Department of Cardio-Pulmonary Circulation, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China Thrombosis and Vascular Medicine Center, State Key Laboratory of Cardiovascular Disease, Fu Wai Hospital, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing, China
a r t i c l e
i n f o
Article history: Received 15 September 2014 Accepted 23 September 2014 Available online 2 October 2014 Keywords: 15-F2t-IsoP Idiopathic pulmonary arterial hypertension Prognosis Biomarkers Preanalytical and analytical
From the authors: We are deeply grateful to Dr Monneret and colleagues for thoughtful comments and appreciate the great interest in our recent article on plasma 15-F2t-isoprostane as a potential biomarker in idiopathic pulmonary arterial hypertension [1]. In the comment letter, the authors propose and discuss the methodology used for the quantification of such a promising biomarker, especially the preanalytical and analytical points [2]. Firstly, we have to admit that the healthy individuals and patients for the measurement of 15-F2t-isoprostane were collected at the same period, preanalytical steps and storage conditions. There are different blood collected proposals in our center according to research projects, i.e. EDTA, heparin and sodium citrate tube. Plasma or serum was divided into aliquots equally at low temperature followed by freezing at − 80 °C until they were analyzed. For oxidant test, the presence of 0.005% BHT (without GSH) was allowed anti-ex-vivo oxidation and improving stability, routinely. As for a retrospective study, a majority of patients were enrolled at 2009–2010, excepting for 5% patients at 2007–2008. It seemed that plasma samples would be stable at least 3 years followed by taking appropriate measurements [3]. Secondly, since a large percentage of 15-F2t-isoprostane may be esterified in lipids within samples, we performed a hydrolyzed procedure ⁎ Corresponding author. Tel.: +86 10 88396018; fax: +86 10 88396016. E-mail address: [email protected] (Z.-C. Jing).
http://dx.doi.org/10.1016/j.ijcard.2014.09.122 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.
using potassium hydroxide (KOH). To test for interference, dilute one or two test samples to obtain at least two different dilutions of each sample to fall with 20–80% B/B0 (Blank/Maximum Binging) for the assay being used. Two different dilutions of the sample (1:20 and 1:50) were preanalyzed in our study as recommended by manufacturers and referred by purification of isoprostanes from plasma samples [4]. Although there was a good correlation (differ less 20%) in the final calculated concentration, we also purified samples by modified solid phase extraction (SPE) cartridges protocol, for reducing inconsistent results, as elsewhere done as we have known. Thus, here we emphasize that multistep sample purification is still a necessary step prior to enzyme immunoassay (EIA) despite that it was complicated and time consuming. Despite the increasing knowledge of accuracy and advantage of the liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS) method, EIA techniques offer ease of performance and are available to measure 15-F2t-IsoPs by commercial vendors [5,6]. A study by Klawitter et al. showed that the EIA tested overestimated 15F2t-isoprostane concentrations in the plasma compared to the corresponding concentrations measured by LC/LC–MS/MS [7]. Relatively, even the overestimation of 15-F2t-isoprostane concentrations, the differences seem to be insistent and accessible in inter-batch and inter-operator variability. As it was a retrospective cohort study, the possibility of sampling selection could be calibrated within a shorter time once follow-up period is blocked. In this regard, we tested over 250 samples (80 controls, 80 patients and 71 follow-up) in triplicate using 3 big packets (4 plates each packet). As quality control, samples with %B/B0 values greater than 80% or less than 20% should be reassayed, and the total activity (TA) values are used as a diagnostic tool. The mean intra-patient coefficient of variation (CV) in baseline and follow-up patients were 21% [10%–27%] and 22% [18%–25%], respectively, which seemed to be acceptable and reasonable. Although this is in agreement with the sensitivity and specificity of LC–ESI-MS, the decision using analytical method would have to be decided by familiarity, cost and personal preference [8]. It also enables quantitative determination of isoprostane regioisomers that are biomarkers of in vivo exposure to oxidative stress, the decision using urine, plasma and exhaled breath condensate. Before selecting or developing a particular analytical method, the investigator is wise to carefully check the published literatures that describe application of existing analytical methods to similar projects.
R. Zhang, Z.-C. Jing / International Journal of Cardiology 177 (2014) 632–633
There is an increasingly intense scientific and clinical interest in oxidative stress and the many parameters used to quantify the degree of oxidative stress. In the future, when analytical limitations of biomolecules arise, optimal strategy and standardization of the methods should be given great importance. Conflict of interest The author/s report/s no relationships that could be construed as a conflict of interest. References [1] Zhang R, Sun ML, Fan YF, Jiang X, Zhao QH, He J, et al. Plasma 15-F2t-isoprostane in idiopathic pulmonary arterial hypertension. Int J Cardiol 2014;175:268–73. [2] Monneret D, Cracowski JL, Bonnefont-Rousselot D. Isoprostane as a promising prognostic biomarker in pulmonary arterial hypertension: preanalytical and analytical viewpoints. Int J Cardiol 2014; Aug 23;177:527–8.
633
[3] Morrow JD, Roberts II LJ. Mass spectrometric quantification of F2-isoprostanes in biological fluids and tissues as measure of oxidant stress. Methods Enzymol 1999;300: 3–12. [4] Lee CY, Huang SH, Jenner AM, Halliwell B. Measurement of F2-isoprostanes, hydroxyeicosatetraenoic products, and oxysterols from a single plasma sample. Free Radic Biol Med 2008;44:1314–22. [5] Lee CYJ, Jenner AM, Halliwell B. Rapid preparation of human urine and plasma samples for analysis of F2-isoprostanes by gas chromatography–mass spectrometry. Biochem Biophys Res Commun 2004;320:696–702. [6] Chu X, Ageishi Y, Nishimura K, Jisaka M, Nagaya T, Shono F, et al. Development of enzyme-linked immunosorbent assay for 8-iso-prostaglandin F2alpha, a biomarker of oxidative stress in vivo, and its application to the quantification in aged rats. J Pharm Biomed Anal 2009;50:911–6. [7] Klawitter J, Haschke M, Shokati T, Klawitter J, Christians U. Quantification of 15-F2tisoprostane in human plasma and urine: results from enzyme-linked immunoassay and liquid chromatography/tandem mass spectrometry cannot be compared. Rapid Commun Mass Spectrom 2011;25:463–78. [8] Janicka M, Kot-Wasik A, Paradziej-Łukowicz J, Sularz-Peszyńska G, Bartoszek A, Namieśnik J. LC–MS/MS determination of isoprostanes in plasma samples collected from mice exposed to doxorubicin or tert-butyl hydroperoxide. Int J Mol Sci 2013; 14:6157–69.
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Letter to the Editor
Isoprostane as a promising biomarker in pulmonary arterial hypertension: Preanalytical and analytical viewpoints. Response to letter to the editor Rui Zhang a, Zhi-Cheng Jing b,⁎ a b
Department of Cardio-Pulmonary Circulation, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China Thrombosis and Vascular Medicine Center, State Key Laboratory of Cardiovascular Disease, Fu Wai Hospital, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing, China
a r t i c l e
i n f o
Article history: Received 15 September 2014 Accepted 23 September 2014 Available online 2 October 2014 Keywords: 15-F2t-IsoP Idiopathic pulmonary arterial hypertension Prognosis Biomarkers Preanalytical and analytical
From the authors: We are deeply grateful to Dr Monneret and colleagues for thoughtful comments and appreciate the great interest in our recent article on plasma 15-F2t-isoprostane as a potential biomarker in idiopathic pulmonary arterial hypertension [1]. In the comment letter, the authors propose and discuss the methodology used for the quantification of such a promising biomarker, especially the preanalytical and analytical points [2]. Firstly, we have to admit that the healthy individuals and patients for the measurement of 15-F2t-isoprostane were collected at the same period, preanalytical steps and storage conditions. There are different blood collected proposals in our center according to research projects, i.e. EDTA, heparin and sodium citrate tube. Plasma or serum was divided into aliquots equally at low temperature followed by freezing at − 80 °C until they were analyzed. For oxidant test, the presence of 0.005% BHT (without GSH) was allowed anti-ex-vivo oxidation and improving stability, routinely. As for a retrospective study, a majority of patients were enrolled at 2009–2010, excepting for 5% patients at 2007–2008. It seemed that plasma samples would be stable at least 3 years followed by taking appropriate measurements [3]. Secondly, since a large percentage of 15-F2t-isoprostane may be esterified in lipids within samples, we performed a hydrolyzed procedure ⁎ Corresponding author. Tel.: +86 10 88396018; fax: +86 10 88396016. E-mail address: [email protected] (Z.-C. Jing).
http://dx.doi.org/10.1016/j.ijcard.2014.09.122 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.
using potassium hydroxide (KOH). To test for interference, dilute one or two test samples to obtain at least two different dilutions of each sample to fall with 20–80% B/B0 (Blank/Maximum Binging) for the assay being used. Two different dilutions of the sample (1:20 and 1:50) were preanalyzed in our study as recommended by manufacturers and referred by purification of isoprostanes from plasma samples [4]. Although there was a good correlation (differ less 20%) in the final calculated concentration, we also purified samples by modified solid phase extraction (SPE) cartridges protocol, for reducing inconsistent results, as elsewhere done as we have known. Thus, here we emphasize that multistep sample purification is still a necessary step prior to enzyme immunoassay (EIA) despite that it was complicated and time consuming. Despite the increasing knowledge of accuracy and advantage of the liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS) method, EIA techniques offer ease of performance and are available to measure 15-F2t-IsoPs by commercial vendors [5,6]. A study by Klawitter et al. showed that the EIA tested overestimated 15F2t-isoprostane concentrations in the plasma compared to the corresponding concentrations measured by LC/LC–MS/MS [7]. Relatively, even the overestimation of 15-F2t-isoprostane concentrations, the differences seem to be insistent and accessible in inter-batch and inter-operator variability. As it was a retrospective cohort study, the possibility of sampling selection could be calibrated within a shorter time once follow-up period is blocked. In this regard, we tested over 250 samples (80 controls, 80 patients and 71 follow-up) in triplicate using 3 big packets (4 plates each packet). As quality control, samples with %B/B0 values greater than 80% or less than 20% should be reassayed, and the total activity (TA) values are used as a diagnostic tool. The mean intra-patient coefficient of variation (CV) in baseline and follow-up patients were 21% [10%–27%] and 22% [18%–25%], respectively, which seemed to be acceptable and reasonable. Although this is in agreement with the sensitivity and specificity of LC–ESI-MS, the decision using analytical method would have to be decided by familiarity, cost and personal preference [8]. It also enables quantitative determination of isoprostane regioisomers that are biomarkers of in vivo exposure to oxidative stress, the decision using urine, plasma and exhaled breath condensate. Before selecting or developing a particular analytical method, the investigator is wise to carefully check the published literatures that describe application of existing analytical methods to similar projects.
R. Zhang, Z.-C. Jing / International Journal of Cardiology 177 (2014) 632–633
There is an increasingly intense scientific and clinical interest in oxidative stress and the many parameters used to quantify the degree of oxidative stress. In the future, when analytical limitations of biomolecules arise, optimal strategy and standardization of the methods should be given great importance. Conflict of interest The author/s report/s no relationships that could be construed as a conflict of interest. References [1] Zhang R, Sun ML, Fan YF, Jiang X, Zhao QH, He J, et al. Plasma 15-F2t-isoprostane in idiopathic pulmonary arterial hypertension. Int J Cardiol 2014;175:268–73. [2] Monneret D, Cracowski JL, Bonnefont-Rousselot D. Isoprostane as a promising prognostic biomarker in pulmonary arterial hypertension: preanalytical and analytical viewpoints. Int J Cardiol 2014; Aug 23;177:527–8.
633
[3] Morrow JD, Roberts II LJ. Mass spectrometric quantification of F2-isoprostanes in biological fluids and tissues as measure of oxidant stress. Methods Enzymol 1999;300: 3–12. [4] Lee CY, Huang SH, Jenner AM, Halliwell B. Measurement of F2-isoprostanes, hydroxyeicosatetraenoic products, and oxysterols from a single plasma sample. Free Radic Biol Med 2008;44:1314–22. [5] Lee CYJ, Jenner AM, Halliwell B. Rapid preparation of human urine and plasma samples for analysis of F2-isoprostanes by gas chromatography–mass spectrometry. Biochem Biophys Res Commun 2004;320:696–702. [6] Chu X, Ageishi Y, Nishimura K, Jisaka M, Nagaya T, Shono F, et al. Development of enzyme-linked immunosorbent assay for 8-iso-prostaglandin F2alpha, a biomarker of oxidative stress in vivo, and its application to the quantification in aged rats. J Pharm Biomed Anal 2009;50:911–6. [7] Klawitter J, Haschke M, Shokati T, Klawitter J, Christians U. Quantification of 15-F2tisoprostane in human plasma and urine: results from enzyme-linked immunoassay and liquid chromatography/tandem mass spectrometry cannot be compared. Rapid Commun Mass Spectrom 2011;25:463–78. [8] Janicka M, Kot-Wasik A, Paradziej-Łukowicz J, Sularz-Peszyńska G, Bartoszek A, Namieśnik J. LC–MS/MS determination of isoprostanes in plasma samples collected from mice exposed to doxorubicin or tert-butyl hydroperoxide. Int J Mol Sci 2013; 14:6157–69.