Gut 1996; 38: 591-597

Faecal phytic acid and its relation to otherputative markers of risk for colorectal cancer

RW Owen, UM Weisgerber, B Spiegelhalder, H Bartsch

AbstractAims-Phytic acid, a major constituent ofcereals, pulses, and seeds has been advo-cated as an important antioxidant compo-nent of dietary fibre that affords possibleprotection against colorectal cancer. Thisis supported by experimental studiesshowing it has antineoplastic activity inanimal models of both colon and breastcancer. To date the concentration offaecal phytic acid in human clinicalgroups has not been evaluated. Thereforethe faecal phytic acid content of adenomapatients drawn from a placebo controlledcalcium intervention trial was evaluated.Methods-Phytic acid was measured infaecal extracts by an improved ion-pairhigh performance liquid chromatographymethod.Results-Phytic acid was detected in therange 0*68-4-00 ,umol/g wet faeces and55-2038 pmollday. Linear regressionanalyses showed no association betweenstool phytic acid and lipid content. Strongcorrelations were seen, however, betweenphytic acid and iron content, both on aconcentration (r=0.52; p=0.00004) anddaily excretion (r=0.76; p=5.5 X10-2)basis. Phytic acid was also strongly corre-lated with the daily excretion of calcium(r=0.59; p=1.36X10-6) and magnesium(r=0.42; p=0.001). Cell proliferation inthe sigmoid colon, an intermediatebiomarker of colorectal cancer was notsignificantly associated with faecal phyticacid, minerals or lipid content in thiscompromised clinical group.Conclusions-This improved method,developed for the determination of phyticacid in faeces should allow further studieson the role of phytic acid in the aetiologyof colorectal cancer to be conducted on apopulation or case control basis.(Gut 1996; 38: 591-597)

Keywords: phytic acid, minerals, lipids, cellproliferation, colorectal cancer, high performanceliquid chromatography.

Phytic acid (PA), a hexaphosphorylated sugar,is a ubiquitous plant component, which mayconstitute 1-5% by weight of most cereals,nuts, legumes, and oil seeds.1 Its presence inseeds is believed to be the reason for theirremarkable longevity. The seeds of someplants may remain viable for up to 400 years.2Graf and Eaton3 suggested that a major reasonfor this is that PA maintains iron in the Fe (III)oxidation state and obstructs generation of

reactive oxygen species, for example, thehydroxyl radical (HO) thereby preventingoxidative damage, especially of unsaturatedfatty acids, which are a major component ofseeds.The antioxidative properties of PA are

ascribed to its chelating potential. Phytic acidexhibits a high affinity for all polyvalent cationsand metal phytate complexes are well knownto be insoluble over a wide pH range and this isparticularly true for iron-phytate chelates.3 Forthis reason Graf and Eaton3 propose that all ofthe antioxidant properties ofPA derive from itscomparatively high binding affinity for iron.Iron catalysed reactions play a crucial part inoxidative damage to biological materials andcan be generated by two commonly describedmechanisms namely the Haber-Weiss andFenton reactions.

For iron to participate in Fenton chemistryit must be chelated and soluble. The six co-ordination sites of trivalent iron, for example,FeCl3.6H2O are occupied by water andhydroxide ions in aqueous solution.4 Mostchelating agents displace five of these ligandsand form a pentadentate chelate with H20occupying the sixth coordination site.4 EDTAforms a hexadentate chelate but because of itssmall size distorts the chelate and makes aseventh site available for H20.4 Phytic acidapparently is unique in occupying six co-ordination sites and displacing all of the co-ordination water in the Fe-III-phytatecomplex.4 The lack of an aquo-coordination.site has been shown spectrophotometricallywith azide and confirmed by nuclear magneticresonance determination.4 Therefore it hasbeen proposed that PA preserves the solubilityof iron making the metal totally unreactive.This is the presumed basis of the antioxidantcapacity of PA and led Graf and Eaton5 tosuggest that the PA content of the diet may beimportant in the modulation of colorectalcancer. Colorectal cancer is a disease ofaffluent societies and epidemiologicalstudies6-8 have clearly shown that dietarycomponents are an important contributoryfactor. High consumption of fat and red meatare regarded important in the promotion ofcolorectal cancer perhaps through an additiveeffect on intestinal metabolism by increasedhepatic secretion of biliary components andfermentation by the intestinal microflora.9 10By contrast high consumption of dietary fibreis regarded ameliorative.'1 12 The protectiverole of dietary fibre against colorectal cancerhas its origins in the classic study of Burkitt.13This was based on epidemiological studies inAfrica, which showed that a high intake ofdietary fibre was highly correlated with faecal

Divisions ofToxicology and CancerRisk FactorsRW OwenB SpiegelhalderH Bartsch

and EpidemiologyU M Weisgerber

German CancerResearch Centre,Heidelberg, GermanyCorrespondence to:Dr R W Owen, Division ofToxicology and Cancer RiskFactors, German CancerResearch Centre, ImNeuenheimer Feld 280,D-69120 Heidelberg,Germany.Accepted for publication31 October 1995

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weight. It was proposed that dietary fibre isprotective against colorectal cancer by thesimple mechanisms of stool bulking, accelera-tion of transit, and dilution of potentialendogenous carcinogens. This theory hasrecently received qualified support in the meta-analyses ofHowe et a114 and Cummings et al.15Nevertheless certain inconsistencies in thedietary fibre data indicate that it may prove

fruitful to analyse the PA content of dietaryfibre and assess its effect on intestinal bio-chemistry.The influence of PA in the genesis of colo-

rectal cancer has received substantial supportfrom animal model studies. Nielson et al16studied the effect of adding PA at varying con-

centrations to the normal diet of the rat. Phyticacid at concentrations of 1.2% and 2% signifi-cantly lowered colonic cell proliferation, an

intermediate biomarker of colorectal cancer inthese rats. Shamsuddin et al 17-19 have shownin a series of studies utilising the azoxymethanerodent model that addition of PA (1-2%) tothe diet significantly reduces tumour burdenand volume. The anticancer effect of PA was

found to be dose dependent and temporal inthat it significantly reduced the incidence andcolon tumour volume when given five monthsafter the last injection of azoxymethane.Thompson and Zhang20 have shown that

PA can reduce cell proliferation in both colonicand mammary tissues in the mouse andfurthermore subverts the promotional effect ofadded iron and calcium. Nelson et al 21 studiedthe combined effects of iron and PA and whilethe first of these was shown to promote 1,2-dimethylhydrazine induced colorectal cancer

in the rat these effects were nullified by simul-taneous administration of the second.

Furthermore iron enriched diets caused an

increase of tumour rate in two models of 1,2-dimethylhydrazine induced colon tumori-genesis in mice.22 23 The effect was

independent of the time the diet was fed, was

dose dependent, and increased both tumourinitiation and promotion.

Further support for the hypothesis of Grafand Eaton comes from studies on iron statusand risk of cancer in humans. Stevens et a124reported that increased body iron stores were

significantly associated with colon cancer in a

national survey (NHANES 1) and this hasbeen supported by a recent case controlstudy25 showing that adenoma risk is positivelyassociated with increased serum ferritin con-

centrations.Despite all the circumstantial evidence, to

date, no study has been conducted in theclinical domain to bring together the variousfactors of the phytic acid/reactive oxygenspecies hypothesis3 and its implications forcancer. Therefore cell proliferation rate, faecallipid, mineral and PA content of patients withsporadic adenomatous polyps have beenmeasured to establish any interrelation thatmay have a bearing on the aetiology of colo-rectal cancer.A modified high performance liquid chro-

matography (HPLC) system was developedand used for PA analyses in selected foods and

human faeces because of the unsatisfactorybehaviour of those already published.

Methods

Chemicals and reagentsPhytic acid (40°/O in water), formic acid,tetrabutylammonium hydroxide (40% inwater), tetrabutylammonium sulphate, andFeCl3.6H20 were obtained from AldrichChemicals (Steinheim, Germany). Potassiumand sodium phytate were obtained from SigmaChemicals (Deisenhofen, Germany). Thesodium salts of inositol hexaphosphate andpentaphospate were obtained from Calbio-chem, La Jolla, California. All solutions weremade up in millipore grade water.

Study groupThe study group comprised 29 adenomapatients (Table I) drawn from a longtermplacebo controlled calcium intervention study(unpublished data).

Faecal samplesFaecal samples were defecated into plasticbowls and placed immediately on dry icebefore transportation to the laboratory. Theywere stored at -80°C until further analyses.For aliquoting, samples were thawed overnightat 4°C, pooled, and homogenised (room tem-perature) at 600 rpm for five minutes in arotating homogeniser (Heidolph, Kelheim,Germany). Aliquots of 40 g were freeze dried,reweighed, and ground to a fine homogeneouspowder by mortar and pestle before the analy-ses of PA, lipids, and minerals. Remainingaliquots were refrozen at -80°C.

Food samplesThe following foods were included in the study- namely, sesame seeds, wheat bran, peanuts,soy beans, coriander seeds, chilli seeds, pepperseeds, tomato seeds, and millett and wereanalysed after being lypholysed, pulverised bymortar and pestle to a fine homogeneouspowder, and defatted by Soxhlet extraction foreight hours essentially as described by Faddenand Owen26 except that chloroform/methanolwas replaced by pentane.

TABLE I Age, sex, and dietary data before intervention inthe study groups

Placebo group Calcium group(n= 15) (n= 15)

Age 63-3 (6(1) 60-6 (9.2)No of female patients 10 4Dietary intake*Energy (kJId) 10410 (2169) 11 678 (5788)Protein (g/d) 93 (27) 99 (50)Fat (g/d) 97 (38) 105 (50)Carbohydrates (g/d) 253 (52) 293 (160)Fibre (g/d) 31 (7) 35 (20)Calcium (mg/d) 933 (416) 922 (370)Vitamin D (gg/d) 4 (4) 4 (3)

*As determined by food frequency questionnaire beforeintervention (adjusted for sex). Results expressed as mean (SD).

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| Faeces (500 mg) |

0.5 M HCI at 25°Cv for two hours

Freeze overnight and centrifuge slurry at 5000 x gfor 20 minutes

Evaporate to dryness

Dissolve in 0.01 M HCI (20 ml)

; 0-4 mI/min

7/ 2.0 g (AG 1-X8,200-400 mesh)

* Discard eluateWith 20 x 1 mi 2 M HCI

Elute inositol phosphates

Evaporate to dryness, freeze-dry, and resuspend indistilled water

HPLCFigure 1: Protocolfor the analysis ofphytic acid.

Sample extraction and preparationSamples in the range 250-1000 mg (routinely250 mg) were extracted (Fig 1) with 20 ml 0.5M HCI under vigorous mechanical agitation(500 rpm) for two hours at room temperature.27The extracts were frozen overnight, thawed,centrifuged at 5000 rpm, and the supernatantswere evaporated to dryness at 65°C. The driedextracts were resuspended in 20 ml 0.01 M HCIand percolated over Dowex anion-exchange gel(1 X 8, 200-400 mesh; 2 g) in glass columns at a

rate of 1 ml/minute. The columns were washedwith a further 40 ml 0.01 M HCI before elutionof PA with 20 ml 2 M HCl at 04 ml/min. Theeluates were dried, resuspended in 20 mldistilled water, filtered through MF-Milliporefilters (0.2 gm), and redried. After freeze-dryingovernight the extracts were resuspended in1.0 ml distilled water.

StandardsA stock PA solution was made by diluting 40°/OPA (Aldrich Chemical) in distilled water togive final concentrations in the range (1-60p,mol/ml). The linearity of PA concentrationversus peak area was investigated by duplicate20 ,u injections of each dilution. For compari-son, standard curves were prepared withsodium phytate and potassium phytate (SigmaChemical). The sodium salts of inositol pen-taphosphate and hexaphosphate were alsostudied in this system.A mixture of inositol phosphates was pre-

pared by autoclaving PA (20 ml) at 121 C forone hour and refluxing sodium phytate (1 g) in100 ml 0.5 M HCI for 16 hours. In the lattercase, samples (5.0 ml) were removed periodi-cally to assess the degradation pattern. Thesamples were evaporated to dryness, freeze-dried, and resuspended in distilled waterbefore injection into the HPLC.

HPLC analysisThree different methods described in pub-lished works were compared for HPLC analy-ses of PA in biological material. The methodsof Graf and Dintzis,27 Sandberg andAhderrine,28 and Lee and Abendroth29 wereapplied exactly as described. For this study amodification of the method of Lee andAbendroth29 was finally adopted as describedbelow.The mobile phase consisted of 0.005 M

tetrabutylammonium hydroxide in distilledwater, pH 4.3 (with formic acid, 6%).Chromatography was conducted on a Hewlett-Packard HP 1090 liquid chromatograph fitted(routinely) with a C-18 (Latek, Eppelheim,Germany) reverse phase 25 cm, (5 ,u) column(internal diameter; ID, 4.6 mm) or else a C-18(Supelco, Bad Homburg, Germany) reversephase 25 cm (5 ,u) column (ID, 2-1 mm).Twenty gl of sample was injected into theHPLC and PA was detected at 210 nm using adiode-array detector at room temperature. Theoptimal flow rate of the mobile phase was1-0 ml/min and the column void volume wasdetermined by injecting NaCl (20 pumolml)dissolved in mobile phase. Instrument controland data handling was by means of a HPChemStation operating in the MicrosoftWindows software environment.

Methods for determining labelling index, faecallipids, and mineralsLabelling index was measured as describedpreviously by Weisgerber et a13' while faecallipids and minerals were analysed by themethods of Owen et al.32-34

Statistical analysesThe interactions between PA, lipids, andminerals in faecal extracts were evaluated bymultiple linear regression.

ResultsThree different methods for the analysis of PAin biological material by HPLC were appraised.The method of Graf and Dintzis27 was found tobe reproducible but as stated by the authors, PAeluted in the void volume and was prone to con-tamination by salts. Furthermore the PA peakwas difficult to integrate because of consider-able vacancy peaks at both the leading and trail-ing edges. The method of Sandberg andAhderrine28 on the other hand was found to becompletely irreproducible. Using the exact con-ditions described PA, sodium phytate, potas-sium phytate, inositol pentaphosphate, andinositol hexaphosphate all eluted in the columnvoid volume. This was the case when eithera 4-6 or 2-1 mm (ID) C-18 column wasused. Various permutations of the conditionsdescribed by Sandberg and Ahderrine28 such asvarying the pH, the proportion of methanol andtetrabutylammoniun hydroxide, substitution oftetrabutylammonium hydroxide with tetra-butylammonium sulphate had no effect onthe retention time: PA in its various forms and

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1400

12000)(0c 100000.au 800

o 60000)gD 4000

200

Phytic acid (RT = 3.70 min)

Void volume(RT = 2.60 min)

11111111 11111111111111111111 liii

0 2 4 6 8 10 12Retention time (min)

14 16 18

Figure 2: HPLC chromatogram of authentic phytic acid.

20 000

0)enC 15 0000

0)h- 10 0000

° 50000)

0

Phytic acid

0 10 20 30 40 50 60 70Phytate (gmol/mI)

Figure 3: Phytate standard curves determined by HPLC.

inositol pentaphosphate always eluted in thevoid volume. The chromatography ofPA in thissystem seems to be heavily dependent on thepresence of methanol. When it was omittedfrom the mobile phase the PA became irretriev-ably bound to the column and could only beremoved by the addition of methanol, whichincidentally also simultaneously co-eluted tetra-butylammonium hydroxide making quantifica-tion ofPA impossible.When the method of Lee and Abendroth29

was used, PA was well separated from the voidvolume but peak shape was extremely poor. Inthis case variation of the conditions gave fruit-ful results. Stepwise lowering of the pH inincrements of 0.5 units improved peak shapeconsiderably with a simultaneous decrease inretention time. Optimum peak shape wasobtained between pH 4-5 and the final pHchosen was 4-3. Under the HPLC conditionsdescribed the void volume eluted at 2.6minutes and PA at 3.7 minutes (Fig 2). Themethod was extremely reproducible and PAconcentration versus peak area in the range

TABLE III Faecal characteristics of the study group

Faecal excretion

Faecal parameter 1Lmollg wetfaeces mmollday

Phytic acid 1-74 (0.69) 0-36 (0.19)Iron 2-03 (0.93) 0.49 (0.49)Calcium

Total 155 (108) 34 (27)Soluble 19 (12) 4-46 (3.58)Soaps 31 (31) 6-85 (7.02)

Magnesium 74 (35) 15 (11)Phosphate 125 (45) 26 (13)Bile acids 4-52 (2.60) 0 94 (0.60)Long chain fatty acids 50 (30) 11 (11)Neutral sterols 11 (6) 2-71 (3.37)Plant sterols 3-23 (2 92) 0-68 (0.72)pH 6-55 (0.42) NALabelling index (/) 11-86 (3.99) NAFaecal wet weight (g) NA 225 (107)

Data expressed as mean (SD) (n=29). NA=not applicable.

1-60 pumol/ml was found to be linearly propor-tional over the entire range (Fig 3). The peakareas of sodium and potassium phytate (Fig 3)and sodium inositol pentaphosphate andinositol hexaphosphate gave similar linearfunctions but of a lesser magnitude because ofa lower proportional content of actual PA. ThePA and sodium phytate digests showed thatthe lower inositol phosphates, like authenticinositol pentaphosphate could not be sepa-rated from PA or inositol hexaphosphate inthis system. This method therefore groups allthe inositol phosphates as PA and gives a valuefor total phytate in biological samples com-pared with the published method of Sandbergand Ahderrine,28 which described earlier couldnot be reproduced.

Analysis ofPA in selected foodstuffs showeda good correlation with other methods butin general the values obtained were lower(Table II) when calculated against free PA asexternal standard. When calculation was madeagainst a sodium phytate standard curve (withcorrection for sodium content), however, thedata were very close to previously publishedvalues. The data show a similar trend to previ-ous studies in that the highest PA content ofthose compared was found in wheat bran andthe lowest in soy beans. Phytic acid concentra-tions were also determined in foodstuffs, whichhave not been previously reported and of thesecoriander, tomato, and green pepper seedswere found to contain appreciable amounts(Table II). Efficiency of extraction was depen-dent on the foodstuff to solvent ratio anddecreased significantly (r=-0 97; p=0.03)

TABLE II Phytic acid (%, dry weight) content ofa range offoods

External standardtValuesfrom

Food sample* Phytic acid Sodium phytate Sodium phytat4 published reports

Sesame seeds 2-21 (0.02) 7-83 (0.07) 5-56 (0.05) 5-36 (0.09)§Wheat bran 2-28 (0.08) 8-14 (0-37) 5-78 (0.22) 5-03 (1.85)11Peanuts 0-86 (0.02) 3.05 (0.07) 2-17 (0.04) 1-88 (0 05)Soy beans 0-89 (0.06) 3.09 (0 22) 2-20 (0-16) 1-84 (0.03)§Tomato seeds 1-24 (0-14) 4-36 (0.49) 3-10 (0.35) NPChilli seeds 0-56 (0.06) 1.90 (0-21) 1-34 (0-15) NPCoriander seeds 1.11 (0.03) 3-93 (0 10) 2-79 (0.07) NPPepperseeds 0-57 (0-01) 1-96 (0.04) 1-39 (0.03) NPMillet 0-18 (001) 0-62 (003) 044 (002) NP

*Analyses conducted as described in Methods. tResults expressed as % (mean (SD)) of two tofour samples: lyophilised, pulverised, and defatted with pentane (not corrected for defatting).*Sodium phytate corrected for salt content. Values from §Graf and Dintzis27 and IlCamire andClydesdale.30 NP=not published.

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a

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Phytic acid (gmol/g wet weight)Figure 4: Correlation between faecal phytic acid and ironexcretion.

nI

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3000

2500

>i 2000

0E 1500C

0 1000

r=0.76;p=5.5x 10-12a

E

0 500 1000 1500 2000Phytic acid (lmol/day)

Figure 5: Correlation between daily faecal phytic acid andiron excretion.

between 1:80 and 1:20 as determined with aconcentration range of defatted sesame seeds(data not shown).The HPLC method was also found to be

appropriate for the measurement of PA inhuman faeces. The acid extracts of the ade-noma patients contained appreciable amountsof proteinacous material (X maximum 240-280 nm as determined by HPLC) but this wasremoved completely by the prior anion-exchange chromatography step.

Phytic acid was detected in faecal extractsof the adenoma patients in the range 0.68-4.00 pumolg wet faeces and 55-2038 pumolday(Table III). There was little difference inthe mean (SD) excretion of PA in theplacebo (1.88 (0.80) v 1.78 (067) pumo1/g)and calcium (1.60 (0.59) v 1-86 (0.88)pumoVg) arms of the composite groups bothbefore and after intervention. Linear regressionanalyses of PA versus faecal lipid and mineralcontent and intestinal cell proliferation (TableIII) showed the following. The amount of PAin the stool was strongly correlated (Fig 4) withfaecal iron (r=0.52; p=000004), unsaturatedfatty acids (r=0.35; p=0.004), and total cal-cium content of the stool (r=0.34; p=0 01). Aweak positive association was seen between PAand magnesium (r=0.26; p=0.04). The asso-ciation between PA and minerals was evenstronger when analysed on a daily basis: PA viron, r=0-76; p=5.5X 10-12 (Fig 5): PA v totalcalcium, r=0-59; p=1'36x10-6 (Fig 6): PA vmagnesium, r=0.42; p=0001. No associationexisted between stool PA content and the

160 - r= 0.59; p =1.36 x 10

140

E 120

E 100 EEUE 80 *

E 60 -'UI

40 -o ~

0 500 1000 1500 2000Phytic acid (,umol/day)

Figure 6: Correlation between daily faecal phytate andcakcium excretion.

40 r

0EEE._n

0Cuc_

10

r= 0-56; p = 5-84 x 10Aa

- m/*L a

A %:a

0 20 40 60 80 100 120 140 160Calcium (mmol/day)

Figure 7: Correlation between dailyfaecal magnesium andcakium excretion.

concentration and daily excretion of phos-phate, bile acids, long chain fatty acids, neutralsterols, and plant sterols.

Intermineral positive associations were alsoseen. The faecal excretion of calcium and mag-nesium was positively correlated on both aconcentration (r=0.36; p=0006) and daily(r=0.56; p=5.84X 10-6) basis (Fig 7).

Cell proliferation, an intermediate bio-marker of colorectal cancer showed no associa-tion with faecal PA (r=0. 17; p=0 20) or withfaecal iron (r=-0.18; p=0.17). It was, how-ever, positively correlated with faecal mag-nesium (r=0-28; p=O004) concentration. Cellproliferation was not correlated with faecaltotal calcium concentration taking the studygroup as a whole (r=0.05; p=0.69) or whencomparison was adjusted for calcium interven-tion, but, was positively correlated with solublecalcium (r=0.30; p=0.03). Faecal soluble cal-cium (Fig 8) was found to be strongly associ-ated with total calcium concentration (r=0 5 1;p=000005). Cell proliferation did not corre-late with the faecal concentration of phos-phate, bile acids, long chain fatty acids,neutrals sterols or calcium-long chain fattyacid soaps and on a daily basis no associationapart for magnesium (r=0-30; p=003) wasfound with any of the faecal characteristics.Furthermore there was no association betweenthe faecal concentration of bile acids in relationto long chain fatty acids and neutral sterols.

DiscussionA reproducible HPLC method has been devel-oped and validated that enables the accuratequantification of PA in faeces. The method,

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0o10 100 200 300 400 500

Total calcium (jimol/g)Figure 8: Correlation between faecal soluble calcium andtotal calcium.

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based on that described by Lee andAbendroth,29 is not over complex and has beensuccessfully applied to the analyses of PA infoodstuffs and faecal specimens in patientswith sporadic adenoma. The method gives avalue for total PA in biological material and asyet cannot be used to resolve the lower inositolphosphates, for example, inositol triphosphate-inositol pentaphosphate from PA as describedby Sandberg and Ahderrine.28 Despite rigor-ous adherence to the method conditionsdescribed by this last group, it could not bereproduced, and all attempts to modify it werein vain. When methanol was omitted from themobile phase, PA was not eluted after severalhours. Addition of methanol up to 55% eitheras increments (5%), or as a linear gradientremoved PA from the column but it always co-eluted with salts and tetrabutylammoniumhydroxide. The crux of the problem here isthat in the presence of methanol, tetrabutylam-monium hydroxide is unable to bind to C-18reverse-phase columns and ion-pair chromato-graphy in this system is impossible to achieve.The reasons for this discrepancy with the pub-lished method of Sandberg and Ahderrine28remain in part unexplained.

Authentic free PA was well resolved fromthe void volume with excellent peak shapeusing the current method. Free PA in compari-son to potassium phytate gave rise to a smallpeak in the void volume while potassiumphytate gave a significant void volume peakdue to dissociation of K+ and PA in the mobilephase. Sodium phytate and sodium inositolpentaphosphate and hexaphosphate eluted inthe void volume and dissociation and resolu-tion of the PA from the void volume could onlybe achieved by a prior anion-exchangechromatography step. Probably the sodiumsalts (Na12) of PA and the lower inositol phos-phates when solubilised in mobile phase atpH 4.3 are not dissociated and therefore PAcannot participate in ion-pair chromatography.How successful HPLC of authentic sodiumsalts of PA and its derivatives has beenachieved as described by various groupsremains unexplained because if PA does notdissociate from the salt then ion-pair chroma-tography with tetrabutylammonium hydroxideis not possible.The PA content of various foodstuffs

obtained in this system compares favourablywith other published methods. The valuesobtained were in general a little lower but asthe comparable published values wereobtained by the HPLC system of Graf andDintzis27 and iron precipitation methodsperhaps this is not surprising.When the method was applied to the analy-

sis of faeces from adenoma patients PA wasdetected in the range 0-68-40 pumolg wetmaterial (55-2038 ,uwmol/day) with little differ-ence in mean values both before and afterintervention. This range when compared on adry weight basis is roughly comparable to thelimited examples published for human and ratfaeces.35

This shows that the subjects included in thishigh risk study group consumed considerable

amounts of PA, because it is generally regardedthat the faecal profile is an adequate mirror ofthis for the following reasons. Studies inhumans and pigs show that approximately60-70% of ingested PA passes undergraded tofaeces after gastrointestinal transit despite indi-cations that some foodstuffs36 and the smallintestine37 of humans and rats contain appre-ciable phytase activity.The data showed a striking correlation

especially on a daily basis between the faecalcontent ofPA and iron, vindicating the hypoth-esis that PA may be important in binding of thision and preventing its absorption and possibleparticipation in reactive oxygen species generat-ing mechanisms in the body. The notion thathigh consumption of PA may contribute toexcessive mineral loss38 was supported by thefact that it was positively associated with faecalexcretion of calcium and magnesium. This wasparticularly true for calcium when adjustmentwas made for calcium intervention.Much of the circumstantial evidence and

especially that obtained from animal modelstudies shows that PA through its ability tochelate iron could play an important part in thechemoprevention of colorectal cancer. There-fore it was important to assess faecal PAcontent in relation to cell proliferation of theintestinal mucosa. However, no associationbetween cell proliferation and stool PA andiron on both a concentration and daily excre-tion basis was seen. It should be noted, how-ever, that in this clinical study the early eventsof colonic neoplasia had already occurred (thatis, adenoma formation) and it would have beenunlikely that stool parameters such as PA andiron concentration would have displayedcorrelations with cell proliferation becausethe presence of adenomas is known to haveconsiderable influence on cell turnover in thecolorectum.39

In conclusion a method has been developedfor the reliable determination of PA in humanfaeces. Stool PA content shows a strong corre-lation with the excretion of minerals such asiron, calcium, and magnesium but exhibits norelation with cell proliferation in this clinicalgroup. Similar studies on a population andcase control basis are warranted, however, tofully assess the role ofPA in colorectal carcino-genesis.

1 Maga JA. Phytate; its chemistry, occurrence, food inter-actions, nutritional significance and methods of analysisJ3Agric Food Chem 1982; 30: 1-9.

2 Ohlrogge JB, Kernan TP. Oxygen dependent ageing ofseeds. Plant Physiol 1982; 70: 791-4.

3 Graf E, Eaton JW. Antioxidant functions of phytic acid. FreeRadic Biol Med 1990; 8: 61-9.

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