- Email: [email protected]
Letter to the Editor concerning Baecher et al. (Clin Chim Acta 2014;431:87–92)
CCA-14040; No of Pages 2 Clinica Chimica Acta xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Letter to the Editor Letter to the Editor concerning Baecher et al. (Clin Chim Acta 2014;431:87–92) Keywords: Endogenous Ouabain Plasma Mass spectroscopy
To the Editor: I write in reference to the Clin Chim Acta article [1] that tests whether the cardiotonic steroid, ouabain, is endogenous in human plasma. This is clinically important. Baecher et al. [1] cite early analytic evidence for endogenous ouabain (EO) in human plasma [2–4], and state that, “increased plasma ouabain was reported in several (human) pathological conditions such as hypertension… and heart failure”. Nevertheless, they claim that “in none of the 30 human plasma samples (they tested) ouabain could be detected” [1]. Serious flaws in their article, however, raise doubt about the validity of their claim. Most important is apparently biased data selection. In Fig. 2 of [1], the mass spectrometry (MS) spectrum in the lower panel (“threefold deuterated IS”, internal standard) is continuous. In contrast, the upper trace (“target analyte ouabain” spectrum) is clearly discontinuous, but this is not mentioned. For example, there appears to be a peak at 5.00 min that has been cut off just above the base. This and several (but not all) other gaps in the upper trace are indicated by arrows in the enlarged segment of Fig. 2 in [1] (here designated Fig. 1L). How do the authors know that the apparently deleted signals (e.g., at 5.00 min), with the same mass/charge ratio as ouabain (perhaps an ouabain isomer; e.g., [5,6]), are unimportant? The upper panel of Fig. 2 [1] shows a plasma sample ‘spiked’ with ouabain, thereby obscuring the signal from the spectrum at the position at which endogenous ouabain (EO) would be expected (5.30 min). Crucially the article shows no original MS spectra from plasma without added analyte (ouabain). How can anyone conclude that there was no ouabain present in the original spectra? Was this not the central issue in the study? Moreover, Dr. Baecher sent 3 plasma samples to Dr. P. Manunta (Milan, Italy) to extract and test for EO by radioimmunoassay (RIA); EO levels of 206–665 pM were detected in those samples (P. Manunta, personal communication, with permission). This was not acknowledged in [1], which discounts all EO RIAs (Table 1 in [1]) even though the ouabain RIA used in Milan and Baltimore has been validated by multi-dimensional MS standardized with ouabain [5] and dihydroouabain [6]. No original ultra-performance liquid chromatography (UPLC) or tandem MS/MS data are shown in [1]. How could this have been overlooked? The absence of UPLC data, needed to demonstrate the efficiency of ouabain extraction during purification of samples for MS, is even more critical than the questionable MS/MS data. Ouabain has
been analytically measured by MS since the 1970s [7]. Hence, MS assay should be routine for experienced mass spectrometrists, even with less sensitive instruments than the one employed in [1]. The demonstration of an ouabain-like substance in human plasma and its purification by liquid chromatography, however, hinges on careful step-by-step verification with multiple assays including binding to the native receptor, the Na,K-ATPase [3]. No such data are shown in [1]. The MS/MS results are irrelevant if EO was lost in the solid phase extraction and UPLC steps. Published HPLC data and MS spectra demonstrating that EO is present in human plasma [2–4,8] will not disappear because of [1]. Importantly, key data from bovine tissue and from rodents, including mice with mutated (resistant) Na,K-ATPase ouabain binding sites (e.g. [11, 12]) were ignored in [1]. Those studies involve: i) MS and NMR spectra of EO (e.g. [5,6,9,10]); and ii) unequivocal (analytical) evidence for EO and for its physio-/pathophysiological roles in mammals [5,6]. Further,
Fig. 1. L. Enlargement of a portion of the spectrum from human EDTA plasma spiked with ouabain (Fig. 2 from [1]); arrows added to indicate gaps (upper panel). The gap at 5.00 min appears to be the position of a peak that has be cut off at the base. There are no gaps in the spectrum of the standard (lower panel) which contained 3-fold deuterated (d3) ouabain. Abscissa units = minutes; ordinate units are arbitrary (%), with the ouabain and ouabaind3 peaks ≈ 100%.
http://dx.doi.org/10.1016/j.cca.2015.07.011 0009-8981/© 2015 Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: M.P. Blaustein, Letter to the Editor concerning Baecher et al. (Clin Chim Acta 2014;431:87–92), Clin Chim Acta (2015), http://dx.doi.org/10.1016/j.cca.2015.07.011
2
Letter to the Editor
mutant mouse studies (e.g. [11,12]) are consistent with earlier reports on the role of EO in human cardiovascular diseases (e.g., references 21–28 in [1]). Of course, this endogenous ligand might have been lost during the evolution of humans while its high affinity receptor site, present in all human cells, was conserved throughout mammalian evolution. More likely, however, the methodological steps in [1] were not all optimized to assure the retention and measurement of EO. The potential clinical relevance is obvious. Given the “objective (of Clin Chim Acta) to publish novel information leading to a better understanding of biological mechanisms of human diseases, their prevention, diagnosis, and patient management”, it seems especially important for the journal to get the story ‘correct’. Surely, ‘proof’ that human plasma does not contain ouabain requires far more than an undocumented statement [1], especially when three published reports with MS spectra [4,5,8] and RIA of plasmas provided by Baecher et al. show that human plasma does contain ouabain. Mordecai P. Blaustein, M.D. References [1] S. Baecher, M. Kroiss, M. Fassnacht, M. Vogeser, No endogenous ouabain is detectable in human plasma by ultra-sensitive UPLC-MS/MS, Clin. Chim. Acta 431 (2014) 87–92. [2] J.H. Ludens, M.A. Clark, D.W. DuCharme, et al., Purification of an endogenous digitalislike factor from human plasma for structural analysis, Hypertension 17 (1991) 923–929. [3] W.R. Mattews, D.W. DuCharme, J.M. Hamlyn, et al., Mass spectral characterization of an endogenous digitalislike factor from human plasma, Hypertension 17 (1991) 930–935. [4] J.M. Hamlyn, M.P. Blaustein, S. Bova, et al., Identification and characterization of a ouabain-like compound from human plasma, Proc. Natl. Acad. Sci. U. S. A. 88 (1991) 6259–6263. [5] B.E. Jacobs, L. Liu, M.V. Pulina, V.A. Golovina, J.M. Hamlyn, Normal pregnancy: mechanisms underlying the paradox of a ouabain-resistant state with elevated endogenous
[6]
[7]
[8]
[9] [10] [11]
[12]
ouabain, suppressed arterial sodium calcium exchange and low blood pressure, Am. J. Physiol. Heart Circ. Physiol. 302 (2012) H1317–H1329. J.M. Hamlyn, C.I. Linde, J. Gao, et al., Neuroendocrine humoral and vascular components in the pressor pathway for brain angiotensin II: a new axis in long term blood pressure control, PLoS One 9 (2014) e108916. J. Vine, L. Brown, J. Bougagy, R. Thomas, D. Nelson, Cardenolide analogues: 10—characterization of cardiac glycosides by chemical ionization mass spectrometry, Biomed. Mass Spectrom. 6 (1979) 415–421. Y. Komiyama, N. Nishimura, X.H. Dong, et al., Liquid chromatography mass spectrophotometric analysis of ouabain-like factor in biological fluid, Hypertens. Res. 23 (2000) S21–S27 (Suppl.). R. Schneider, V. Wray, M. Nimtz, et al., Bovine adrenals contain, in addition to ouabain, a second inhibitor of the sodium pump, J. Biol. Chem. 273 (1998) 784–792. A. Kawamura, J. Guo, Y. Itagaki, et al., On the structure of endogenous ouabain, Proc. Natl. Acad. Sci. U. S. A. 86 (1999) 6654–6659. J.N. Lorenz, E.L. Loreaux, I. Dostanic-Larson, et al., ACTH-induced hypertension is dependent upon the ouabain-binding site of the α2 Na+–K+-ATPase subunit, Am. J. Physiol. Heart Circ. Physiol. 295 (2008) H273–H280. A.N. Wansapura, V.M. Laszko, J.B. Lingrel, J.N. Lorenz, Mice expressing ouabainsensitive α1-Na, K-ATPase have increased susceptibility to pressure overloadinduced cardiac inotropy, Am. J. Physiol. Heart Circ. Physiol. 300 (2010) H347–H355.
Mordecai P. Blaustein Department of Physiology, University of Maryland School of Medicine, Baltimore, MD, USA Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, USA Department of Physiology, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, MD 21201, USA. E-mail address: [email protected]. 17 June 2015 Available online xxxx
Please cite this article as: M.P. Blaustein, Letter to the Editor concerning Baecher et al. (Clin Chim Acta 2014;431:87–92), Clin Chim Acta (2015), http://dx.doi.org/10.1016/j.cca.2015.07.011
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Letter to the Editor Letter to the Editor concerning Baecher et al. (Clin Chim Acta 2014;431:87–92) Keywords: Endogenous Ouabain Plasma Mass spectroscopy
To the Editor: I write in reference to the Clin Chim Acta article [1] that tests whether the cardiotonic steroid, ouabain, is endogenous in human plasma. This is clinically important. Baecher et al. [1] cite early analytic evidence for endogenous ouabain (EO) in human plasma [2–4], and state that, “increased plasma ouabain was reported in several (human) pathological conditions such as hypertension… and heart failure”. Nevertheless, they claim that “in none of the 30 human plasma samples (they tested) ouabain could be detected” [1]. Serious flaws in their article, however, raise doubt about the validity of their claim. Most important is apparently biased data selection. In Fig. 2 of [1], the mass spectrometry (MS) spectrum in the lower panel (“threefold deuterated IS”, internal standard) is continuous. In contrast, the upper trace (“target analyte ouabain” spectrum) is clearly discontinuous, but this is not mentioned. For example, there appears to be a peak at 5.00 min that has been cut off just above the base. This and several (but not all) other gaps in the upper trace are indicated by arrows in the enlarged segment of Fig. 2 in [1] (here designated Fig. 1L). How do the authors know that the apparently deleted signals (e.g., at 5.00 min), with the same mass/charge ratio as ouabain (perhaps an ouabain isomer; e.g., [5,6]), are unimportant? The upper panel of Fig. 2 [1] shows a plasma sample ‘spiked’ with ouabain, thereby obscuring the signal from the spectrum at the position at which endogenous ouabain (EO) would be expected (5.30 min). Crucially the article shows no original MS spectra from plasma without added analyte (ouabain). How can anyone conclude that there was no ouabain present in the original spectra? Was this not the central issue in the study? Moreover, Dr. Baecher sent 3 plasma samples to Dr. P. Manunta (Milan, Italy) to extract and test for EO by radioimmunoassay (RIA); EO levels of 206–665 pM were detected in those samples (P. Manunta, personal communication, with permission). This was not acknowledged in [1], which discounts all EO RIAs (Table 1 in [1]) even though the ouabain RIA used in Milan and Baltimore has been validated by multi-dimensional MS standardized with ouabain [5] and dihydroouabain [6]. No original ultra-performance liquid chromatography (UPLC) or tandem MS/MS data are shown in [1]. How could this have been overlooked? The absence of UPLC data, needed to demonstrate the efficiency of ouabain extraction during purification of samples for MS, is even more critical than the questionable MS/MS data. Ouabain has
been analytically measured by MS since the 1970s [7]. Hence, MS assay should be routine for experienced mass spectrometrists, even with less sensitive instruments than the one employed in [1]. The demonstration of an ouabain-like substance in human plasma and its purification by liquid chromatography, however, hinges on careful step-by-step verification with multiple assays including binding to the native receptor, the Na,K-ATPase [3]. No such data are shown in [1]. The MS/MS results are irrelevant if EO was lost in the solid phase extraction and UPLC steps. Published HPLC data and MS spectra demonstrating that EO is present in human plasma [2–4,8] will not disappear because of [1]. Importantly, key data from bovine tissue and from rodents, including mice with mutated (resistant) Na,K-ATPase ouabain binding sites (e.g. [11, 12]) were ignored in [1]. Those studies involve: i) MS and NMR spectra of EO (e.g. [5,6,9,10]); and ii) unequivocal (analytical) evidence for EO and for its physio-/pathophysiological roles in mammals [5,6]. Further,
Fig. 1. L. Enlargement of a portion of the spectrum from human EDTA plasma spiked with ouabain (Fig. 2 from [1]); arrows added to indicate gaps (upper panel). The gap at 5.00 min appears to be the position of a peak that has be cut off at the base. There are no gaps in the spectrum of the standard (lower panel) which contained 3-fold deuterated (d3) ouabain. Abscissa units = minutes; ordinate units are arbitrary (%), with the ouabain and ouabaind3 peaks ≈ 100%.
http://dx.doi.org/10.1016/j.cca.2015.07.011 0009-8981/© 2015 Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: M.P. Blaustein, Letter to the Editor concerning Baecher et al. (Clin Chim Acta 2014;431:87–92), Clin Chim Acta (2015), http://dx.doi.org/10.1016/j.cca.2015.07.011
2
Letter to the Editor
mutant mouse studies (e.g. [11,12]) are consistent with earlier reports on the role of EO in human cardiovascular diseases (e.g., references 21–28 in [1]). Of course, this endogenous ligand might have been lost during the evolution of humans while its high affinity receptor site, present in all human cells, was conserved throughout mammalian evolution. More likely, however, the methodological steps in [1] were not all optimized to assure the retention and measurement of EO. The potential clinical relevance is obvious. Given the “objective (of Clin Chim Acta) to publish novel information leading to a better understanding of biological mechanisms of human diseases, their prevention, diagnosis, and patient management”, it seems especially important for the journal to get the story ‘correct’. Surely, ‘proof’ that human plasma does not contain ouabain requires far more than an undocumented statement [1], especially when three published reports with MS spectra [4,5,8] and RIA of plasmas provided by Baecher et al. show that human plasma does contain ouabain. Mordecai P. Blaustein, M.D. References [1] S. Baecher, M. Kroiss, M. Fassnacht, M. Vogeser, No endogenous ouabain is detectable in human plasma by ultra-sensitive UPLC-MS/MS, Clin. Chim. Acta 431 (2014) 87–92. [2] J.H. Ludens, M.A. Clark, D.W. DuCharme, et al., Purification of an endogenous digitalislike factor from human plasma for structural analysis, Hypertension 17 (1991) 923–929. [3] W.R. Mattews, D.W. DuCharme, J.M. Hamlyn, et al., Mass spectral characterization of an endogenous digitalislike factor from human plasma, Hypertension 17 (1991) 930–935. [4] J.M. Hamlyn, M.P. Blaustein, S. Bova, et al., Identification and characterization of a ouabain-like compound from human plasma, Proc. Natl. Acad. Sci. U. S. A. 88 (1991) 6259–6263. [5] B.E. Jacobs, L. Liu, M.V. Pulina, V.A. Golovina, J.M. Hamlyn, Normal pregnancy: mechanisms underlying the paradox of a ouabain-resistant state with elevated endogenous
[6]
[7]
[8]
[9] [10] [11]
[12]
ouabain, suppressed arterial sodium calcium exchange and low blood pressure, Am. J. Physiol. Heart Circ. Physiol. 302 (2012) H1317–H1329. J.M. Hamlyn, C.I. Linde, J. Gao, et al., Neuroendocrine humoral and vascular components in the pressor pathway for brain angiotensin II: a new axis in long term blood pressure control, PLoS One 9 (2014) e108916. J. Vine, L. Brown, J. Bougagy, R. Thomas, D. Nelson, Cardenolide analogues: 10—characterization of cardiac glycosides by chemical ionization mass spectrometry, Biomed. Mass Spectrom. 6 (1979) 415–421. Y. Komiyama, N. Nishimura, X.H. Dong, et al., Liquid chromatography mass spectrophotometric analysis of ouabain-like factor in biological fluid, Hypertens. Res. 23 (2000) S21–S27 (Suppl.). R. Schneider, V. Wray, M. Nimtz, et al., Bovine adrenals contain, in addition to ouabain, a second inhibitor of the sodium pump, J. Biol. Chem. 273 (1998) 784–792. A. Kawamura, J. Guo, Y. Itagaki, et al., On the structure of endogenous ouabain, Proc. Natl. Acad. Sci. U. S. A. 86 (1999) 6654–6659. J.N. Lorenz, E.L. Loreaux, I. Dostanic-Larson, et al., ACTH-induced hypertension is dependent upon the ouabain-binding site of the α2 Na+–K+-ATPase subunit, Am. J. Physiol. Heart Circ. Physiol. 295 (2008) H273–H280. A.N. Wansapura, V.M. Laszko, J.B. Lingrel, J.N. Lorenz, Mice expressing ouabainsensitive α1-Na, K-ATPase have increased susceptibility to pressure overloadinduced cardiac inotropy, Am. J. Physiol. Heart Circ. Physiol. 300 (2010) H347–H355.
Mordecai P. Blaustein Department of Physiology, University of Maryland School of Medicine, Baltimore, MD, USA Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, USA Department of Physiology, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, MD 21201, USA. E-mail address: [email protected]. 17 June 2015 Available online xxxx
Please cite this article as: M.P. Blaustein, Letter to the Editor concerning Baecher et al. (Clin Chim Acta 2014;431:87–92), Clin Chim Acta (2015), http://dx.doi.org/10.1016/j.cca.2015.07.011