Journal of Chromatography B, 960 (2014) 255–257
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Letter to the Editor Phytate levels in biological fluids of mammals Keywords: Phytate levels Myo-inositol hexaphosphate Biological fluids
Dear Editor, We have read Prof. Irvine’s letter with great interest, commenting on our recent publication in this journal [1]. We would like to thank him in advance for his interest in this fascinating field and in our paper; however, in this response letter we will explain our concerns in relation to his conclusions regarding the absence of detectable myo-inositol hexaphosphate (IP6, phytate) in biological fluids. Certainly, the bioanalysis of IP6 is a complex issue. In spite of being discovered during the second half of the 19th century, its physiological role has been progressively understood in parallel with the development of appropriate analytical tools for its bioanalytical determination. An important limitation is the fact that IP6 does not significantly absorb light from any region of the UV–visible spectrum; however, several analytical methodologies have been successfully developed and applied for its quantification in simple matrices, but the number of described applications for urine and plasma is rather limited, because of the complexity of the media in which it can be found. It is necessary to distinguish between intracellular and extracellular IP6. Intracellular IP6 are relatively high, in the 10–100 M range [2–4]. There seems to be a general consensus that extracellular levels are much lower, typically in the 0.5–3 M for urine and 0.1–0.3 M for plasma. That includes data from our research group [5–7], but it is important to consider that independent authors have confirmed these results [8–11], and this fact highlights the controversial findings described by Letcher et al. [12], which have not been follow-up by confirming studies by either themselves or other research groups. In their publication, Letcher et al. [12] state that there is no detectable IP6 in human and urine plasma, basing their conclusions in the analysis of one single urine sample and three blood samples. It is remarkable that in the thousands of urines and hundreds of blood samples we have analyzed during the last 17 years we have found some humans and rats having undetectable, probably depending on their diet and/or other unknown aspects. Assuming that, by chance, that might happen with the analysis of 1 urine and 3 blood samples, that is not incompatible with the fact that pre-analytical sample treatment is one of the key issues when determining IP6 specially in blood and Letcher et al. [12] might have lost IP6 during the blood samples pre-treatment. In our research (unpublished data) we noticed that around 1 M IP6 is lost in this process unless EDTA is used as anticoagulant in whole blood. The use of citrate or heparin in this step led to
1570-0232/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.12.016
poor or null recoveries. Since in his letter Prof. Irvine admits that in the recovery studies IP6 was spiked in plasma (not in whole blood), the loss of natural IP6 in the previous step (blood centrifugation) is likely. In addition, we are aware that enzymatic assays for IP6 are tricky, since phosphatases (phytases) and kinases often show high substrate (IP6) levels which render high backgrounds in the assays; this usually does not impact on determinations in the 10–100 M range (i.e. tissues, culture cells) but impairs quantification of urine and blood samples; however, this is just speculation without having more detailed information from their methodology. During our track record, we have developed several methodologies to quantify IP6 in biological samples (urine and blood), all of them based on different indirect approaches: hydrolysis followed by phosphate determination [13], hydrolysis followed by inositol determination [14,15], IP6 isolation and quantification through phosphorus levels by atomic emission spectroscopy [16,17]. Our results have been confirmed by other authors using ICP-MS [8,9] or fluorescence techniques [10,11]. Of course, there are other clinical evidences and experiments whose results only can be explained admitting the presence in plasma and urine of IP6 of dietary origin. Thus, the ingestion of IP6 through the diet increases the inhibitory capacity of urine against calcium oxalate crystallization [18]. A large study with stone-formers demonstrated that the consumption of IP6 decreases in a 36% the possibility to develop calcium stones [19]. How is it possible to explain that this substance in not found in urine of rats fed with a rat-chow without IP6 (AIN-76A diet), and when IP6 is added to that diet then IP6 appeared in urine [20]? How is it possible to explain that the size of induced subepithelial calcifications was clearly and significantly reduced when IP6 was added to the AIN-76A diet [21]? These are only some representative examples of the mentioned evidences. In our recent publication in this journal [1] we presented a direct determination of IP6 in blood. Prof Irvine argues that our chromatograms representing un-spiked samples show other compounds at the m/z 659; of course, it is usual when working with LC–MS and biological samples in SIM mode, since the selectivity is based on retention time and molecular weight. However, as described in our paper, we used 2 different equipments. A UPLC–MS/MS equipment (Waters, Acquity) with an AB Sciex, API4000 mass spectrometer and a HPLC apparatus (Waters, Alliance 2795HT® ) coupled to a ZQ 4000TM (Waters) mass spectrometer. In a further optimization and revalidation of our methodology, we confirmed the specificity of our assay by working with the MRM (Multiple Reaction Monitoring9 mode, so the IP6 presence if confirmed not only by retention time and molecular weight, but also through fragmentation pattern. In Fig. 1 it can be seen how the same un-spiked dog plasma sample is analyzed with the same API4000 equipment, showing a cleaner chromatogram with the MRM mode (compared to the SIM mode) that unquestionably confirms the natural presence of IP6.
256
Letter to the Editor / J. Chromatogr. B 960 (2014) 255–257
Finally, Prof Irvine questions our estimation of IP6 endogenous levels, since we report a lower limit of quantification (LLOQ) higher than these estimated levels. As explained in the paper, our assay was validated according to the FDA text: Bioanalytical Method Validation [22]. So, we have to differentiate between analytical and regulatory sensitivity. The strict rules that apply in
drug development lead to very strict criteria that made us fix the regulatory LLOQ at 500 ng/ml (not 500 nM as Prof. Irvine states in his letter). However, the analytical sensitivity is much higher and as explained in our paper, the analytical limit of detection (not quantification) is in the 30–80 ng/ml range; so, based on all the previous explanations we can assure that the analyzed rat, dog
Fig. 1. (a) Chromatogram obtained for a blank dog plasma sample in a SIM mode, the peak at 3.44 min showed the presence of endogenous IP6 in plasma. Phytic acid retention time 3.44 min. (b) Chromatogram obtained for a blank dog plasma sample in a MRM mode, the peak at 4.20 min showed the presence of endogenous IP6 in plasma. Phytic acid retention time 4.20 min. (c) Chromatogram obtained for the LLOQ (500 ng/mL) in dog plasma sample in a MRM mode. IP6 retention time 4.16 min.
Letter to the Editor / J. Chromatogr. B 960 (2014) 255–257
257
Fig. 1. (Continued ).
and human plasma samples have detectable levels, in the worst of the cases higher than 80 ng/ml (120 nM), thus far in excess of the levels reported by Letcher et al. [12]. To conclude this letter, in contrast to the conclusions drawn by Letcher et al., we firmly believe IP6 is present in plasma in the 0.1–0.3 M range and encourage anyone in this field to perform a previous deep study of the pre-analytical sample treatment and use a wide number of biological samples in order to take firm conclusions.
[17] F. Grases, J. Perello, B. Isern, R.M. Prieto, Anal. Chim. Acta 510 (2004) 41. [18] F. Grases, R. Garcia-Gonzalez, J.J. Torres, A. Llobera, Scand. J. Urol. Nephrol. 32 (1998) 261. [19] G.C. Curhan, W.C. Willett, E.L. Knight, M.J. Stampfer, Arch. Intern. Med. 164 (2004) 885. [20] F. Grases, B.M. Simonet, J.G. March, R.M. Prieto, BJU Int. 85 (2000) 138. [21] F. Grases, J. Perello, R.M. Prieto, B.M. Simonet, J.J. Torres, Life Sci. 75 (2004) 11. [22] Guidance for Industry, Bioanalytical Method Validation, U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM), 2001.
Joan Perelló a,b,∗ Sanifit Laboratoris S.L. Research and Development Department, 07121 Palma de Mallorca, Spain b Laboratory of Renal Lithiasis Research, Institute of Health Sciences Research (IUNICS), University of Balearic Islands, 07122 Palma of Mallorca, Spain a
References [1] F. Tur, E. Tur, I. Lentheric, P. Mendoza, M. Encabo, B. Isern, F. Grases, C. Maraschiello, J. Perelló, J. Chromatogr. B 928 (2013) 146. [2] S.B. Shears, Cell. Signal. 13 (2001) 151. [3] F. Grases, B.M. Simonet, I. Vucenik, J. Perelló, R.M. Prieto, A.M. Shamsuddin, Life Sci. 71 (2002) 1535. [4] J.P. Frederik, D. Mattiske, J.A. Wofford, L.C. Megosh, L.Y. Drake, S.T. Chiou, B.L.M. Morgan, J.D. York, Proc. Natl. Acad. Sci. 102 (2005) 8454. [5] F. Grases, B.M. Simonet, I. Vucenik, R.M. Prieto, Biofactors 15 (2001) 53. [6] F. Grases, B.M. Simonet, R.M. Prieto, J.G. March, Br. J. Nutr. 86 (2001) 225. [7] A.A. López-González, F. Grases, N. Monroy, B. Marí, M.T. Vicente-Herrero, F. Tur, J. Perelló, Eur. J. Nutr. 52 (2013) 717. ˜ [8] J.A. Munoz, M. Valiente, Anal. Chem. 75 (2003) 6374. ˜ [9] J.A. Munoz, M. López-Mesas, M. Valiente, Anal. Chim. Acta 658 (2010) 204. [10] Y. Chen, J. Chen, K. Ma, S. Cao, X. Chen, Anal. Chim. Acta 605 (2007) 185. [11] S. Cao, N. Dong, J. Chen, Phytochem. Anal. 22 (2011) 119. [12] A.J. Letcher, M.J. Schell, R.F. Irvine, Biochem. J. 416 (2008) 263. [13] J.G. March, B.M. Simonet, F. Grases, Anal. Chim. Acta 367 (1998) 36. [14] J.G. March, B.M. Simonet, F. Grases, J. Chromatogr. B 757 (2001) 247. [15] J. Perello, B. Isern, J.A. Munoz, M. Valiente, F. Grases, Chromatographia 60 (2004) 265. [16] F. Grases, A. Llobera, Anal. Lett. 29 (1996) 1193.
Felix Grases Laboratory of Renal Lithiasis Research, Institute of Health Sciences Research (IUNICS), University of Balearic Islands, 07122 Palma of Mallorca, Spain ∗ Corresponding
author at: Sanifit Laboratoris S.L. Research and Development Department, 07121 Palma de Mallorca, Spain. Tel.: +34 971 439 925; fax: +34 871 962 545. E-mail address: joan.perello@sanifit.com (J. Perelló) 4 December 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 Phytate levels in biological fluids of mammals Keywords: Phytate levels Myo-inositol hexaphosphate Biological fluids
Dear Editor, We have read Prof. Irvine’s letter with great interest, commenting on our recent publication in this journal [1]. We would like to thank him in advance for his interest in this fascinating field and in our paper; however, in this response letter we will explain our concerns in relation to his conclusions regarding the absence of detectable myo-inositol hexaphosphate (IP6, phytate) in biological fluids. Certainly, the bioanalysis of IP6 is a complex issue. In spite of being discovered during the second half of the 19th century, its physiological role has been progressively understood in parallel with the development of appropriate analytical tools for its bioanalytical determination. An important limitation is the fact that IP6 does not significantly absorb light from any region of the UV–visible spectrum; however, several analytical methodologies have been successfully developed and applied for its quantification in simple matrices, but the number of described applications for urine and plasma is rather limited, because of the complexity of the media in which it can be found. It is necessary to distinguish between intracellular and extracellular IP6. Intracellular IP6 are relatively high, in the 10–100 M range [2–4]. There seems to be a general consensus that extracellular levels are much lower, typically in the 0.5–3 M for urine and 0.1–0.3 M for plasma. That includes data from our research group [5–7], but it is important to consider that independent authors have confirmed these results [8–11], and this fact highlights the controversial findings described by Letcher et al. [12], which have not been follow-up by confirming studies by either themselves or other research groups. In their publication, Letcher et al. [12] state that there is no detectable IP6 in human and urine plasma, basing their conclusions in the analysis of one single urine sample and three blood samples. It is remarkable that in the thousands of urines and hundreds of blood samples we have analyzed during the last 17 years we have found some humans and rats having undetectable, probably depending on their diet and/or other unknown aspects. Assuming that, by chance, that might happen with the analysis of 1 urine and 3 blood samples, that is not incompatible with the fact that pre-analytical sample treatment is one of the key issues when determining IP6 specially in blood and Letcher et al. [12] might have lost IP6 during the blood samples pre-treatment. In our research (unpublished data) we noticed that around 1 M IP6 is lost in this process unless EDTA is used as anticoagulant in whole blood. The use of citrate or heparin in this step led to
1570-0232/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.12.016
poor or null recoveries. Since in his letter Prof. Irvine admits that in the recovery studies IP6 was spiked in plasma (not in whole blood), the loss of natural IP6 in the previous step (blood centrifugation) is likely. In addition, we are aware that enzymatic assays for IP6 are tricky, since phosphatases (phytases) and kinases often show high substrate (IP6) levels which render high backgrounds in the assays; this usually does not impact on determinations in the 10–100 M range (i.e. tissues, culture cells) but impairs quantification of urine and blood samples; however, this is just speculation without having more detailed information from their methodology. During our track record, we have developed several methodologies to quantify IP6 in biological samples (urine and blood), all of them based on different indirect approaches: hydrolysis followed by phosphate determination [13], hydrolysis followed by inositol determination [14,15], IP6 isolation and quantification through phosphorus levels by atomic emission spectroscopy [16,17]. Our results have been confirmed by other authors using ICP-MS [8,9] or fluorescence techniques [10,11]. Of course, there are other clinical evidences and experiments whose results only can be explained admitting the presence in plasma and urine of IP6 of dietary origin. Thus, the ingestion of IP6 through the diet increases the inhibitory capacity of urine against calcium oxalate crystallization [18]. A large study with stone-formers demonstrated that the consumption of IP6 decreases in a 36% the possibility to develop calcium stones [19]. How is it possible to explain that this substance in not found in urine of rats fed with a rat-chow without IP6 (AIN-76A diet), and when IP6 is added to that diet then IP6 appeared in urine [20]? How is it possible to explain that the size of induced subepithelial calcifications was clearly and significantly reduced when IP6 was added to the AIN-76A diet [21]? These are only some representative examples of the mentioned evidences. In our recent publication in this journal [1] we presented a direct determination of IP6 in blood. Prof Irvine argues that our chromatograms representing un-spiked samples show other compounds at the m/z 659; of course, it is usual when working with LC–MS and biological samples in SIM mode, since the selectivity is based on retention time and molecular weight. However, as described in our paper, we used 2 different equipments. A UPLC–MS/MS equipment (Waters, Acquity) with an AB Sciex, API4000 mass spectrometer and a HPLC apparatus (Waters, Alliance 2795HT® ) coupled to a ZQ 4000TM (Waters) mass spectrometer. In a further optimization and revalidation of our methodology, we confirmed the specificity of our assay by working with the MRM (Multiple Reaction Monitoring9 mode, so the IP6 presence if confirmed not only by retention time and molecular weight, but also through fragmentation pattern. In Fig. 1 it can be seen how the same un-spiked dog plasma sample is analyzed with the same API4000 equipment, showing a cleaner chromatogram with the MRM mode (compared to the SIM mode) that unquestionably confirms the natural presence of IP6.
256
Letter to the Editor / J. Chromatogr. B 960 (2014) 255–257
Finally, Prof Irvine questions our estimation of IP6 endogenous levels, since we report a lower limit of quantification (LLOQ) higher than these estimated levels. As explained in the paper, our assay was validated according to the FDA text: Bioanalytical Method Validation [22]. So, we have to differentiate between analytical and regulatory sensitivity. The strict rules that apply in
drug development lead to very strict criteria that made us fix the regulatory LLOQ at 500 ng/ml (not 500 nM as Prof. Irvine states in his letter). However, the analytical sensitivity is much higher and as explained in our paper, the analytical limit of detection (not quantification) is in the 30–80 ng/ml range; so, based on all the previous explanations we can assure that the analyzed rat, dog
Fig. 1. (a) Chromatogram obtained for a blank dog plasma sample in a SIM mode, the peak at 3.44 min showed the presence of endogenous IP6 in plasma. Phytic acid retention time 3.44 min. (b) Chromatogram obtained for a blank dog plasma sample in a MRM mode, the peak at 4.20 min showed the presence of endogenous IP6 in plasma. Phytic acid retention time 4.20 min. (c) Chromatogram obtained for the LLOQ (500 ng/mL) in dog plasma sample in a MRM mode. IP6 retention time 4.16 min.
Letter to the Editor / J. Chromatogr. B 960 (2014) 255–257
257
Fig. 1. (Continued ).
and human plasma samples have detectable levels, in the worst of the cases higher than 80 ng/ml (120 nM), thus far in excess of the levels reported by Letcher et al. [12]. To conclude this letter, in contrast to the conclusions drawn by Letcher et al., we firmly believe IP6 is present in plasma in the 0.1–0.3 M range and encourage anyone in this field to perform a previous deep study of the pre-analytical sample treatment and use a wide number of biological samples in order to take firm conclusions.
[17] F. Grases, J. Perello, B. Isern, R.M. Prieto, Anal. Chim. Acta 510 (2004) 41. [18] F. Grases, R. Garcia-Gonzalez, J.J. Torres, A. Llobera, Scand. J. Urol. Nephrol. 32 (1998) 261. [19] G.C. Curhan, W.C. Willett, E.L. Knight, M.J. Stampfer, Arch. Intern. Med. 164 (2004) 885. [20] F. Grases, B.M. Simonet, J.G. March, R.M. Prieto, BJU Int. 85 (2000) 138. [21] F. Grases, J. Perello, R.M. Prieto, B.M. Simonet, J.J. Torres, Life Sci. 75 (2004) 11. [22] Guidance for Industry, Bioanalytical Method Validation, U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM), 2001.
Joan Perelló a,b,∗ Sanifit Laboratoris S.L. Research and Development Department, 07121 Palma de Mallorca, Spain b Laboratory of Renal Lithiasis Research, Institute of Health Sciences Research (IUNICS), University of Balearic Islands, 07122 Palma of Mallorca, Spain a
References [1] F. Tur, E. Tur, I. Lentheric, P. Mendoza, M. Encabo, B. Isern, F. Grases, C. Maraschiello, J. Perelló, J. Chromatogr. B 928 (2013) 146. [2] S.B. Shears, Cell. Signal. 13 (2001) 151. [3] F. Grases, B.M. Simonet, I. Vucenik, J. Perelló, R.M. Prieto, A.M. Shamsuddin, Life Sci. 71 (2002) 1535. [4] J.P. Frederik, D. Mattiske, J.A. Wofford, L.C. Megosh, L.Y. Drake, S.T. Chiou, B.L.M. Morgan, J.D. York, Proc. Natl. Acad. Sci. 102 (2005) 8454. [5] F. Grases, B.M. Simonet, I. Vucenik, R.M. Prieto, Biofactors 15 (2001) 53. [6] F. Grases, B.M. Simonet, R.M. Prieto, J.G. March, Br. J. Nutr. 86 (2001) 225. [7] A.A. López-González, F. Grases, N. Monroy, B. Marí, M.T. Vicente-Herrero, F. Tur, J. Perelló, Eur. J. Nutr. 52 (2013) 717. ˜ [8] J.A. Munoz, M. Valiente, Anal. Chem. 75 (2003) 6374. ˜ [9] J.A. Munoz, M. López-Mesas, M. Valiente, Anal. Chim. Acta 658 (2010) 204. [10] Y. Chen, J. Chen, K. Ma, S. Cao, X. Chen, Anal. Chim. Acta 605 (2007) 185. [11] S. Cao, N. Dong, J. Chen, Phytochem. Anal. 22 (2011) 119. [12] A.J. Letcher, M.J. Schell, R.F. Irvine, Biochem. J. 416 (2008) 263. [13] J.G. March, B.M. Simonet, F. Grases, Anal. Chim. Acta 367 (1998) 36. [14] J.G. March, B.M. Simonet, F. Grases, J. Chromatogr. B 757 (2001) 247. [15] J. Perello, B. Isern, J.A. Munoz, M. Valiente, F. Grases, Chromatographia 60 (2004) 265. [16] F. Grases, A. Llobera, Anal. Lett. 29 (1996) 1193.
Felix Grases Laboratory of Renal Lithiasis Research, Institute of Health Sciences Research (IUNICS), University of Balearic Islands, 07122 Palma of Mallorca, Spain ∗ Corresponding
author at: Sanifit Laboratoris S.L. Research and Development Department, 07121 Palma de Mallorca, Spain. Tel.: +34 971 439 925; fax: +34 871 962 545. E-mail address: joan.perello@sanifit.com (J. Perelló) 4 December 2013 Available online 27 December 2013
