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Absence of detectable inositol hexakisphosphate (phytate) in plasma
Journal of Chromatography B, 960 (2014) 253–254
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Letter to the Editor Absence of detectable inositol hexakisphosphate (phytate) in plasma夽
a r t i c l e
i n f o
Keywords: Inositol Inositol phosphates Inositol hexakisphosphate Phytate
a b s t r a c t A critical evaluation of a recent attempt to measure inositol hexakisphosphate (IP6) in mammalian plasma by mass spectroscopy leads to the conclusion that as yet there is no unambiguous evidence that plasma contains any IP6. © 2014 The Author. Published by Elsevier B.V. All rights reserved.
In a recent paper in the Journal of Chromatography B, Tur et al. [1] describe their attempts to use mass spectroscopy to quantify IP6 levels in plasma, and then they discuss the considerable differences between these and the much lower (<0.5 nM) levels that our laboratory reported previously using an enzyme-based bioassay [2]. There appear to be two problems with the way that Tur et al. [1] compare our contrasting sets of data. (1) In their discussion of Ref. [2] Tur et al. do not at any point address scientific issues, but instead rely entirely on (what could be described as inappropriately phrased) rhetoric. For example, they comment in their Introduction [1] that our work [2] is ‘controversial’, and that it ‘erroneously determined’ IP6 levels. This is not an objective scientific argument. Our method is not ‘controversial’ just because it contradicts what the authors and their colleagues claim, and to call something ‘erroneous’ because of that contradiction is not objective but invective. In their Discussion [1] the authors attribute the differences in data to our ‘poor recovery of IP6 as a result of inadequate sample pre-treatment, or inaccurate signal measurement’. There is no space to re-iterate here all the extensive controls and quantifications that we included in our paper [2] – anyone interested should read it and make up their own minds – but in brief, we described there how we did indeed experience problems with loss of IP6 during processing, and how that eventually led us deliberately to keep our sample pre-treatment to the minimum (trichloroacetic acid to precipitate protein and then removal of the acid with ether extraction). This engendered its own problems of interference with the enzyme-based assay (because the more minimal the processing, the more complex the mixture to be analysed), but in Ref. [2] we record in detail how we used numerous IP6-spiked replicates to control fully for any problems resulting from using this simple protocol. The specificity and accuracy of the assay was also
夽 This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike License, which permits noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
validated by analysing a number of eukaryotic tissues for which reliable measurements of IP6 already exist. Moreover, in the most relevant context of human plasma, it is particularly important to stress that, as we also describe clearly and illustrate with raw data (Ref. [2], Fig. 2), some fresh human plasma samples were directly spiked with IP6 (at 25 nM, a concentration lower than that claimed to be present by Tur et al. [1]) before any processing at all. We then showed that this IP6 was, after processing, easily quantified as a large signal (in the example shown in Ref. [2] with only scintillation counter background subtracted: 25 nM IP6-spiked plasma, 3134 dpm; unspiked plasma, 352 dpm; enzyme alone control, 359 dpm;). So in those experiments ‘poor recovery’ is simply not an issue. Similar data were obtained from replicate experiments and also using two other human donors, and overall we reasoned that the upper limit of IP6 in plasma must be about 0.5 nM. Finally, we believe that anyone reading Ref. [2] and noting the extreme care that we took to ensure unambiguous identification and quantification of IP6 throughout would agree that ‘inaccurate signal measurement’ is precisely what it was not. (2) This disappointing lack of discussion of our actual data might be easier to accept if the data presented in Ref. [1] rigorously backed up those authors’ claims. With regard to their own quantification of IP6, the numbers estimated for endogenous levels are lower than the ‘Lower Limit of Quantification’ that they have themselves determined, so it is difficult for the reader to know the linearity at those low levels and so to judge how this quantification was achieved. More importantly, the chromatograms representing un-spiked samples (Ref. [1], Fig. 3) show that, unsurprisingly, a large number of compounds are present in plasma, some at high levels, which give a signal at m/z 659. Yet despite this no data are presented to support the claim that the particular peak that elutes closest to the elution point of IP6 standards actually contains any IP6 at all, nor that if any IP6 is present it is the major contributor to the m/z 659 signal there; these appear simply to be assumptions. Given the chemical complexity of plasma, the increase in any signal that follows resupplying to IP6 to the diet (Ref. [1], Table 3) does not help identification in this context because IP6 is a major source of
1570-0232/$ – see front matter © 2014 The Author. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.12.015
254
Letter to the Editor / J. Chromatogr. B 960 (2014) 253–254
dietary inositol and so its administration would impact directly on all inositol-containing molecules, with consequent indirect effects on the animal’s entire physiology and metabolism (see Ref. [2] for a discussion of this issue). Additional physiological changes will also result from effects on the gut environment caused by the potent polyvalent cation-binding properties of IP6 (also discussed in Ref. [2]). In conclusion, the extensive experiments on recovery and validation of spiked plasma samples reported by Tur et al. in their paper [1] show that the authors can indeed recover exogenous IP6 and quantify it at above 500 nM. But in the absence of either unambiguous data proving that there is endogenous IP6 present in their samples before spiking or any tenable arguments that call our previous data into question, there presently seems to be no reason to modify our original conclusion [2] that there is no (<0.5 nM) IP6 present in mammalian plasma.
References [1] F. Tur, E. Tur, I. Lentheric, P. Mendoza, M. Encabo, B. Isern, F. Grases, C. Marachiello, J. Perelló, J. Chromatogr. B 928 (2013) 146. [2] A.J. Letcher, M.J. Schell, R.F. Irvine, Biochem. J. 416 (2008) 263.
Robin F. Irvine ∗ Department of Pharmacology, Tennis Court Road, Cambridge CB2 1PD, UK ∗ Tel.: +44 1223 334177. E-mail address: rfi[email protected]
24 October 2013 Available online 27 December 2013
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Letter to the Editor Absence of detectable inositol hexakisphosphate (phytate) in plasma夽
a r t i c l e
i n f o
Keywords: Inositol Inositol phosphates Inositol hexakisphosphate Phytate
a b s t r a c t A critical evaluation of a recent attempt to measure inositol hexakisphosphate (IP6) in mammalian plasma by mass spectroscopy leads to the conclusion that as yet there is no unambiguous evidence that plasma contains any IP6. © 2014 The Author. Published by Elsevier B.V. All rights reserved.
In a recent paper in the Journal of Chromatography B, Tur et al. [1] describe their attempts to use mass spectroscopy to quantify IP6 levels in plasma, and then they discuss the considerable differences between these and the much lower (<0.5 nM) levels that our laboratory reported previously using an enzyme-based bioassay [2]. There appear to be two problems with the way that Tur et al. [1] compare our contrasting sets of data. (1) In their discussion of Ref. [2] Tur et al. do not at any point address scientific issues, but instead rely entirely on (what could be described as inappropriately phrased) rhetoric. For example, they comment in their Introduction [1] that our work [2] is ‘controversial’, and that it ‘erroneously determined’ IP6 levels. This is not an objective scientific argument. Our method is not ‘controversial’ just because it contradicts what the authors and their colleagues claim, and to call something ‘erroneous’ because of that contradiction is not objective but invective. In their Discussion [1] the authors attribute the differences in data to our ‘poor recovery of IP6 as a result of inadequate sample pre-treatment, or inaccurate signal measurement’. There is no space to re-iterate here all the extensive controls and quantifications that we included in our paper [2] – anyone interested should read it and make up their own minds – but in brief, we described there how we did indeed experience problems with loss of IP6 during processing, and how that eventually led us deliberately to keep our sample pre-treatment to the minimum (trichloroacetic acid to precipitate protein and then removal of the acid with ether extraction). This engendered its own problems of interference with the enzyme-based assay (because the more minimal the processing, the more complex the mixture to be analysed), but in Ref. [2] we record in detail how we used numerous IP6-spiked replicates to control fully for any problems resulting from using this simple protocol. The specificity and accuracy of the assay was also
夽 This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike License, which permits noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
validated by analysing a number of eukaryotic tissues for which reliable measurements of IP6 already exist. Moreover, in the most relevant context of human plasma, it is particularly important to stress that, as we also describe clearly and illustrate with raw data (Ref. [2], Fig. 2), some fresh human plasma samples were directly spiked with IP6 (at 25 nM, a concentration lower than that claimed to be present by Tur et al. [1]) before any processing at all. We then showed that this IP6 was, after processing, easily quantified as a large signal (in the example shown in Ref. [2] with only scintillation counter background subtracted: 25 nM IP6-spiked plasma, 3134 dpm; unspiked plasma, 352 dpm; enzyme alone control, 359 dpm;). So in those experiments ‘poor recovery’ is simply not an issue. Similar data were obtained from replicate experiments and also using two other human donors, and overall we reasoned that the upper limit of IP6 in plasma must be about 0.5 nM. Finally, we believe that anyone reading Ref. [2] and noting the extreme care that we took to ensure unambiguous identification and quantification of IP6 throughout would agree that ‘inaccurate signal measurement’ is precisely what it was not. (2) This disappointing lack of discussion of our actual data might be easier to accept if the data presented in Ref. [1] rigorously backed up those authors’ claims. With regard to their own quantification of IP6, the numbers estimated for endogenous levels are lower than the ‘Lower Limit of Quantification’ that they have themselves determined, so it is difficult for the reader to know the linearity at those low levels and so to judge how this quantification was achieved. More importantly, the chromatograms representing un-spiked samples (Ref. [1], Fig. 3) show that, unsurprisingly, a large number of compounds are present in plasma, some at high levels, which give a signal at m/z 659. Yet despite this no data are presented to support the claim that the particular peak that elutes closest to the elution point of IP6 standards actually contains any IP6 at all, nor that if any IP6 is present it is the major contributor to the m/z 659 signal there; these appear simply to be assumptions. Given the chemical complexity of plasma, the increase in any signal that follows resupplying to IP6 to the diet (Ref. [1], Table 3) does not help identification in this context because IP6 is a major source of
1570-0232/$ – see front matter © 2014 The Author. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.12.015
254
Letter to the Editor / J. Chromatogr. B 960 (2014) 253–254
dietary inositol and so its administration would impact directly on all inositol-containing molecules, with consequent indirect effects on the animal’s entire physiology and metabolism (see Ref. [2] for a discussion of this issue). Additional physiological changes will also result from effects on the gut environment caused by the potent polyvalent cation-binding properties of IP6 (also discussed in Ref. [2]). In conclusion, the extensive experiments on recovery and validation of spiked plasma samples reported by Tur et al. in their paper [1] show that the authors can indeed recover exogenous IP6 and quantify it at above 500 nM. But in the absence of either unambiguous data proving that there is endogenous IP6 present in their samples before spiking or any tenable arguments that call our previous data into question, there presently seems to be no reason to modify our original conclusion [2] that there is no (<0.5 nM) IP6 present in mammalian plasma.
References [1] F. Tur, E. Tur, I. Lentheric, P. Mendoza, M. Encabo, B. Isern, F. Grases, C. Marachiello, J. Perelló, J. Chromatogr. B 928 (2013) 146. [2] A.J. Letcher, M.J. Schell, R.F. Irvine, Biochem. J. 416 (2008) 263.
Robin F. Irvine ∗ Department of Pharmacology, Tennis Court Road, Cambridge CB2 1PD, UK ∗ Tel.: +44 1223 334177. E-mail address: rfi[email protected]
24 October 2013 Available online 27 December 2013