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Published online 16 May 2007
Published in Soil Sci Soc Am J 71:1045-1050 (2007)
DOI: 10.2136/sssaj2006.0295
© 2007 Soil Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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NUTRIENT MANAGEMENT & SOIL & PLANT ANALYSIS

Identification of Phytate in Phosphorus-31 Nuclear Magnetic Resonance Spectra: The Need for Spiking

Ronald J. Smernik* and Warwick J. Dougherty

Soil and Land Systems, School of Earth and Environmental Sciences, Univ. of Adelaide, Waite Campus Urrbrae, SA, 5064, Australia

* Corresponding author (ronald.smernik{at}adelaide.edu.au).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Phosphorus-31 (31P) nuclear magnetic resonance (NMR) spectroscopy of sodium hydroxide–ethylenediaminetetra-acetic acid (NaOH-EDTA) extracts has recently become a widely used technique for the characterization of soil P. This technique has seemingly enabled easy identification and quantification of phytate (myo-inositol hexakisphosphate), a compound long believed to constitute a major proportion of organic P. Phytate is usually identified by its characteristic pattern of four resonances in the ratio 1:2:2:1. We report that the 31P NMR spectra of the NaOH-EDTA extracts of four Australian pasture soils contain a set of resonances that bear a striking resemblance to the phytate resonances but that are shown not to be phytate though careful addition (spiking) of pure phytate. The spiking experiments identify a much smaller set of resonances as being phytate. Quantification of these resonances shows that phytate comprises <5% of organic P and <3% of total P in these soils. We also show that the 31P chemical shift of phytate resonances is very sensitive to pH and ionic strength. These results highlight the potential for misassignment of resonances in 31P NMR spectra of NaOH-EDTA extracts of soil and the possibility that phytate concentrations may be overestimated using this technique and show the value of spiking as a definitive form of identification of 31P NMR resonances.

Abbreviations: NaOH-EDTA, sodium hydroxide–ethylenediaminetetraacetic acid • NMR, nuclear magnetic resonance • 31P, phosphorus-31


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Phosphorus is an essential plant nutrient. Although the total concentration of P in most soils is more than sufficient for plant nutrition, plant P deficiency often occurs because much of the soil P is not plant available because it is in insoluble inorganic forms and organic P compounds that cannot be used directly by plants. This poor availability has traditionally been addressed by adding inorganic P fertilizers, but this P is often lost through the formation of insoluble inorganic P species (Hedley and McLaughlin, 2005). The organic P pool, which comprises 20 to 80% of P in most soils (Dalal, 1977), has been promoted as an alternative source of plant-available P. In particular, it has been suggested that phytate (myo-inositol hexakisphosphate), widely believed to be an abundant form of organic P in soils (Anderson, 1967; Dalal, 1977; Turner et al., 2002; Turner et al., 2003; McDowell et al., 2005; Smith et al., 2006) but poorly available to plants, could be used as a P source through the insertion of genes into plants that promote extracellular phytase production (Richardson et al., 2001; George et al., 2004).

Phytate was first identified in soil in 1940 (Dyer et al., 1940; Yoshida, 1940) and was reported to represent up to half of the soil organic P pool as early as 1945 (Bower, 1945). A series of methodologies was developed for the quantification of phytate, most involving base extraction, acid or base hydrolysis, and chromatographic isolation (Caldwell and Black, 1958; Cosgrove, 1963; McKercher and Anderson, 1968; Steward and Tate, 1971). In 1981, Irving and Cosgrove reported that some of these methods may overestimate phytate concentrations in soils (Irving and Cosgrove, 1981). Part of the difficulty in determining the concentration of phytate was releasing it from "complexed" forms (Anderson and Hance, 1963; Omotoso and Wild, 1970b; Hong and Yamane, 1981; Borie et al., 1989).

Newman and Tate (1980) reported the first use of phosphorus-31 (31P) nuclear magnetic resonance (NMR) spectroscopy for the characterization of soil P and reported the similarity of prominent resonances in the soil spectra to those of free phytate. In 2003, Turner et al. (2003) described a technique for the quantification of phytate using 31P NMR spectroscopy. This involved the identification of phytate resonances by comparison with the chemical shift of free phytate and subsequent quantification by spectral deconvolution. The relative ease of this technique compared with the older chromatographic techniques has led to its rapid adoption as the preferred technique for quantifying phytate in soils (Chen et al., 2004; McDowell et al., 2005; Smith et al., 2006). In general, results from 31P NMR analysis seem to have supported the case established with the earlier chromatographic techniques that phytate comprises a significant proportion of organic P in most soils and the majority of organic P in some. However, an apparent paradox between the requirement for extensive pretreatment in the older chromatographic techniques to release the phytate from "organic complexes" and the assignment of phytate in 31P NMR spectra in (untreated) soil extracts based on comparison with the 31P spectrum of free phytate has gone unmentioned.

In this article, we re-examine the use of 31P NMR spectroscopy for the identification and quantification of phytate in soil extracts. In particular, we show how careful spiking experiments can be used to identify phytate resonances and describe a novel procedure to quantify the phytate in these soil extracts.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Sodium Hydroxide–Ethylenediaminetetra-acetic Acid Extraction
Standard procedures were used for the extraction of soil organic P with sodium hydroxide–ethylenediaminetetra-acetic acid (NaOH-EDTA) (Cade-Menun and Preston, 1996). This involved shaking 2.5 g of soil with 50 mL of 0.25 M NaOH and 0.05 M Na2–EDTA for 16 h, followed by centrifugation (1300 g) and filtration of the supernatant through a Whatman #42 filter paper. A subsample of the filtered supernatant was analyzed for orthophosphate using the molybdenum blue colorimetric method (Murphy and Riley, 1962). Total P in the supernatant was measured by inductively coupled plasma–atomic emission spectrometer after digestion with nitric acid. The organic P content of the supernatant was estimated as the difference between total P and orthophosphate. The remaining supernatant was frozen and freeze-dried.

Soil Phosphorus Analyses
Total P contents of the whole soils were determined by nitric-perchloric acid digestion (Olsen and Sommers, 1982) and analysis by inductively coupled plasma–atomic emission spectrometer. Organic P contents were determined using the ignition method (Saunders and Williams, 1955).

NMR Analyses
Freeze-dried NaOH-EDTA extracts were ground, and 500 mg was dissolved in 5 mL of deionized water. In all cases, the pH of the resulting solution was >13. The solution was centrifuged (1300 g) for 20 min to remove particles >0.1 µm in diameter. The supernatant solution (3.5 mL) and D2O (0.3 mL) were placed in a 10 mm NMR tube. Solution 31P NMR spectra were acquired at 24°C on a Varian INOVA400 NMR spectrometer (Varian, Palo Alta, CA) at a 31P frequency of 161.9 MHz. Recovery delays range from 15 to 20 s and were set to at least five times the T1 value of the orthophosphate resonance determined in preliminary inversion-recovery experiments. We used a 90° pulse of 60 to 80 µs, an acquisition time of 1.0 s, and broadband 1H decoupling. Chemical shifts were referenced to external 85% H3PO4. The spectra presented have a line broadening of 2 Hz.

Spiking Experiments
Soil NaOH-EDTA extracts (3.5 mL + 0.3 mL D2O) were spiked with 0.2 mL of phytate (sodium salt from corn; Sigma P 8810) solution (269 mg L–1 for Camden, Flaxley and Hamilton extracts; 135 mg L–1 for Waite extract) and 31P spectra reacquired under identical conditions as used for the unspiked extracts.


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
The four soils used in this study are all topsoils from under permanent pasture and represent a range of common soil types found in south-eastern Australia. Sample locations and properties of these soils are detailed in Table 1. Table 2 shows the results of various soil P analyses: total P content of the soils ranged from 545 to 1299 mg kg–1, organic P comprised between 28% and 61% of total P, and between 49% and 60% of the organic P (estimated by ignition) was extracted by NaOH-EDTA using standard methods (Cade-Menun and Preston, 1996).


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  		Table 1. Soil sampling locations, climate and soil properties.

 

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  		Table 2. Soil phosphorus characteristics.

 
The 31P NMR spectra of NaOH-EDTA extracts of the four soils are shown in Fig. 1. The spectra are similar in appearance to previously published 31P NMR spectra of NaOH-EDTA extracts of soils (Chen et al., 2004; Turner and Richardson, 2004; McDowell et al., 2005; McDowell and Stewart, 2006; Smith et al., 2006). The distribution of total 31P NMR signal in diagnostic chemical shift regions for the spectra is presented in Table 3. Orthophosphate represented the majority (51.0–72.0%) of NMR signal intensity (Table 3). Orthophosphate monoesters in the chemical shift region from 5.6 to 3.6 ppm represented between 22.9% and 42.8% of NMR signal intensity (Table 3), and orthophosphate diesters and pyrophosphate each represented no more than 4% of the total signal (Table 3).


Figure 1
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  		Fig. 1. Phosphorus-31 nuclear magnetic resonance spectra of NaOH-EDTA soil extracts. The vertical scale has been increased by a factor of 10 in the inset spectra.

 

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  		Table 3. Percentage of total phosphorus-31 nuclear magnetic resonance signal in diagnostic chemical shift regions.

 
The 31P spectra shown in Fig. 2 (lower trace of each pair) are expansions of the spectra shown in Fig. 1, showing the detail of the orthophosphate and orthophosphate monoester regions. Figure 2 also shows corresponding 31P NMR spectra after the addition of a small amount of phytate (upper traces of each pair). The phytate resonances are easily identified as those that are enhanced in the spiked spectra and are marked with an asterisk at approximately 4.8 to 4.9 ppm, 4.5 ppm, and 4.3 to 4.4 ppm. Another phytate resonance is overlapped by the large orthophosphate resonance and therefore is not visible. It is clear from comparison of the pairs of unspiked and spiked spectra in Fig. 2 that the resonances of the phytate spike do not correspond with the strong resonances present in the 31P NMR spectra of unspiked soil extracts, but rather with minor resonances (i.e., phytate is only a minor component of P in these soils).


Figure 2
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  		Fig. 2. Phosphorus-31 nuclear magnetic resonance spectra of unspiked NaOH-EDTA soil extracts (bottom traces of each pair) and phytate-spiked (top traces) NaOH-EDTA soil extracts. *Phytate resonances. {dagger}Orthophosphate resonance.

 
The usual method for quantifying phytate resonances in 31P NMR spectra is spectral deconvolution (Turner et al., 2003). However, the low intensity of the phytate resonances relative to other adjacent (and sometimes overlapping) resonances (Fig. 2), coupled with the presence of broad underlying signals and a relatively high noise level, made deconvolution unreliable. We therefore used a novel method to determine the concentration of phytate-P in each soil (illustrated in Fig. 3 for the Camden soil) that takes advantage of the fact that the spike produces an authentic spectrum of phytate under the exact conditions present in each extract solution. First, the 31P NMR spectrum of the unspiked extract (Fig. 3a) was subtracted from that of the spiked extract (Fig. 3b) to give the 31P NMR spectrum of the added phytate (Fig. 3c). Increasing proportions of this phytate spectrum (Fig. 3c) were subtracted from the 31P NMR spectrum of the unspiked soil (Fig. 3a) until the phytate resonances in the resultant spectrum were nulled (Fig. 3d). The concentration of phytate-P in the soil extract was calculated from the concentration and volume of phytate solution added and the proportion of the phytate spectrum subtracted. These results are shown in Table 2. Uncertainty in the phytate-P concentration determined in this way stems mainly from uncertainty in the proportion of the phytate spike spectrum required for cancellation of the soil extract phytate resonances. Cancellation of resonances was gauged by eye because the presence of overlapping resonances made it impossible to automate this procedure. Upper and lower limits of phytate-P concentration were determined in a similar way: The lower limit was defined by the maximum proportion of phytate spectrum subtracted that left distinct positive phytate resonances in the resultant spectrum, and the upper limit was defined by the minimum proportion of phytate spectrum subtracted that gave distinct negative phytate resonances in the resultant spectrum. For the Camden and Hamilton soils, for which the phytate resonances are well defined in the 31P NMR spectrum of the unspiked soil extract, this uncertainty is much smaller than for the Flaxley and Waite soils, for which the phytate resonances are much smaller. In fact, for the Waite soil, which has the lowest organic P concentration and hence the poorest spectral signal/noise ratio, the lower bound of phytate-P concentration was zero (i.e., the uncertainty in the phytate-P concentration is 100%).


Figure 3
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  		Fig. 3. Phosphorus-31 (31P) nuclear magnetic resonance (NMR) spectra used to determine the phytate concentration in the Camden soil. (a) 31P NMR spectrum of unspiked NaOH-EDTA extract. (b) Phosphorus 31 (31P) NMR spectrum of NaOH-EDTA extract spiked with phytate. (c) 31P NMR spectrum of phytate spike generated by subtracting a from b. (d) 31P NMR spectrum containing nulled phytate resonances generated by subtracting 0.2 x Spectrum c from Spectrum a.

 
Our calculations showed that phytate-P comprised <5% of organic P and <3% of total P in the soils we examined (Table 2). These phytate-P concentrations are much lower than those reported in a number of recent studies that also used 31P NMR analysis of NaOH-EDTA extracts (Turner et al., 2003; Chen et al., 2004; McDowell et al., 2005; Smith et al., 2006). These differences may be real because soil phytate concentrations are known to vary widely (Turner et al., 2002). However, there is a risk of overestimating phytate concentrations through the misassignment of 31P resonances to phytate.

In previous studies, phytate resonances were not identified by spiking but rather were assigned by comparison with phytate spectra acquired separately or published in the literature (Newman and Tate, 1980; Turner et al., 2003; Chen et al., 2004; McDowell et al., 2005; Smith et al., 2006). The 31P NMR spectra of each of our soil extracts contain a set of strong resonances at around 5.1, 4.7, and 4.6 ppm (Fig. 2) that bear a striking resemblance to three of the phytate resonances: they appear in similar relative proportions to the phytate resonances (2:2:1) and appear consistently just 0.1 to 0.2 ppm to higher chemical shift. Because the chemical shift of phytate resonances varies considerably—three different sets of values reported in the literature vary by 0.5 to 1 ppm (Kemme et al., 1999; Turner et al., 2003; Turner, 2004)—it would be easy to misassign these three strong resonances as phytate. The variation in phytate chemical shifts is further illustrated in Fig. 4, which shows that phytate chemical shifts are strongly affected by changes in pH (even for pH values over 13) and ionic strength. This is problematic for soil extracts because although the pH and ionic strength of the extracting solution can be controlled, the pH and ionic strength of the resulting soil extract is affected by the soil pH and buffering capacity and by the concentration and nature of salts in the soil. The fourth phytate resonance should aid in the identification of phytate because it appears at higher chemical shift (~5.9 ppm) than most other monoesters. This resonance has been used for the quantification of phytate in animal manures (Turner, 2004). This resonance is sometimes overlapped by the much larger orthophosphate resonance (even when pH is >13). This was the case for our soils because only three resonances increased in intensity on spiking (Fig. 2). Presumably, others have inferred this to be the case because high phytate contents have been ascribed to soils whose spectra do not contain prominent signals around 5.9 ppm (Turner et al., 2003; Chen et al., 2004; McDowell et al., 2005; Smith et al., 2006).


Figure 4
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  		Fig. 4. Phosphorus-31 nuclear magnetic resonance spectra of an orthophosphate and phytate mixture at varying pH and ionic strength. {dagger}Orthophosphate resonance.

 
The only certain way to avoid the problem of misassignment is by spiking phytate directly into each soil extract, as we have done. Under these conditions, the added phytate has an identical chemical shift to any phytate that is in solution. Spiking has been used previously to identify phytate in a soil extract (Turner et al., 2003), but in this case a large amount of phytate was added, which may have affected the phytate chemical shifts. We added only a small amount (0.2 mL) of dilute (135 or 269 mgL–1) phytate solution to our soil extracts (3.8 mL). Even so, the orthophosphate resonance shifted slightly (by up to 0.031 ppm) to higher field (lower ppm values) on spiking. The organic P resonances also shifted to higher field but by no more than 0.015 ppm. We limited the amount of phytate added so that the organic P resonances were easily visible in the spiked spectra (Fig. 2b), and hence changes in their chemical shift could be easily detected.

Although phytate has long been believed to be an abundant form of organic P in soils (Anderson, 1967; Dalal, 1977; Turner et al., 2002; Turner et al., 2003; McDowell et al., 2005; Smith et al., 2006), until the introduction of the 31P NMR-based analyses, it was thought that most phytate was present not in the free form but in ill-defined organic complexes (Anderson and Hance, 1963; Omotoso and Wild, 1970b; Hong and Yamane, 1981; Borie et al., 1989). Most chromatographic methods of soil phytate analysis, which predate the 31P NMR method, include pretreatment with hypobromite to destroy these complexes and release the free phytate to solution (Cosgrove, 1963; Omotoso and Wild, 1970a; Omotoso and Wild, 1970b; Irving and Cosgrove, 1981). The similarity of the resonances at 5.1, 4.7, and 4.6 ppm (see Fig. 1) to those of phytate suggests that there may be some connection: These may be due to phytate complexed in some way to organic matter. If these resonances are due to a form of "organically complexed" phytate, any interconversion between the free and complexed forms must be slow because the intensity of these resonances did not increase with the addition of free phytate within the time required for 31P NMR analysis (around 48 h).


   CONCLUSIONS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
This study highlights a potential source of error in the established 31P NMR method used for the identification and quantification of phytate in soils, namely the potential for misassignment of resonances as phytate. It also offers a method for avoiding this problem, namely the definitive identification of phytate via spiking of small amounts of phytate solution into the NaOH-EDTA extracts. In some cases, phytate is readily identifiable and is clearly the major organic P species in NaOH-EDTA extracts, for example in the broiler litter of Turner (2004) and in the Pukaki soil of Chen et al. (2004). In other cases (McDowell et al., 2005; McDowell and Stewart, 2006; Smith et al., 2006), high proportions of organic P are ascribed to phytate based on 31P spectra that are complex and lack an intense signal at ~5.9 ppm. Some doubt must be raised over the phytate concentrations reported in these studies based on the results we report here.

Accurate and reliable identification of organic P compounds is important if organic P is to be better used as a source of plant-available P. The belief that phytate represents a large proportion of organic P in soils has motivated attempts to increase the plant availability of soil organic P through genetic modifications that result in increased production of phytase by plant roots (Richardson et al., 2001; George et al., 2004). Although the P nutrition of such transgenic plants is improved when grown on agar with phytate as a P source (Richardson et al., 2001; George et al., 2004), the transgenic plants do not seem to be able to extract extra P from whole soils (George et al., 2004). This has been attributed to sorption of the phytate (Lung and Lim, 2006) or phytase (George et al., 2005) to the soil mineral phase. However, if free phytate represents only a small proportion of soil P, as is the case for our soils, and perhaps more generally if soil phytate concentrations have been overestimated, then exudation of phytase would not be as beneficial to plant P nutrition as had been envisaged.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
current address: NSW Dep. of Primary Industries, Locked Bag 4, Richmond, NSW, 2753, Australia

All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.

Received for publication August 23, 2006.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 




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