Gut 1994; 35: 1233-1236 1233 Phytase activity in the human and rat small intestine Gut: first published as 10.1136/gut.35.9.1233 on 1 September 1994. Downloaded from

T H Iqbal, K 0 Lewis, B T Cooper

Abstract the enzyme or its distribution in the intestine.5 Phytate is the major storage form ofphos- Metabolic balance studies in human volunteers phorus in seeds and so is a common have suggested that the human intestine is dietary constituent. Excessive ingestion of poor at metabolising phytic acid.9 undegraded phytates can cause mineral Our aim was to measure human intestinal deficiencies in humans. In addition, phytase activity in vitro on fresh small intesti- phytic acid is antineoplastic in animal nal material and to compare this with the models of both colon and breast activity in the rat. As there has been con- carcinoma. There have been no previous troversy in the past as to whether phytase studies quantifying phytase activity in activity actually results from alkaline phos- the human small intestine although it phatase,'0 we planned to measure alkaline is present in animals. Small intestinal phosphatase activity in the same tissues and to phytase and alkaline phosphatase activity use phenyalalanine, which is known to inhibit and distribution was measured in vitro in alkaline phosphatase but not phytase. 1" mucosal homogenates from two human small intestinal specimens obtained from transplant donors. Rat intestine was also Methods studied for comparison. Phytase activity Phytase activity is determined by measuring the was found in human small intestine at liberation of inorganic phosphate when inositol low values (30 times less than that in rat hexaphosphate is broken down. Most methods tissue and 1000-fold lower than alkaline for measuring phosphate are based on the phosphatase in the same tissue). The colour formed by the reduction of phospho- activity was greatest in the duodenum molybdate complexes. 12 Various reducing and lowest in the ileum. In conclusion, agents have been used. The application of this

the normal human small intestine has principle to the determination of phytase http://gut.bmj.com/ very limited ability to digest undegraded activity has been complicated by the finding that phytates. Although this may have adverse phytate itself interferes in these colorimetric nutritional consequences with respect determinations of inorganic phosphate.'3 A to metabolic cation imbalances, the published method for the determination of presence of undigested phytate in the phytase in duodenal biopsy specimens entails colon may protect against the develop- the reduction of phosphomolybdate complexes

ment ofcolonic carcinoma. with ascorbic acid after the extraction of these on September 27, 2021 by guest. Protected copyright. (Gut 1994; 35: 1233-1236) complexes from remaining phytates using isobutyl/heptane.14 In a preliminary trial of this method, Phytate (myo-inositol hexaphosphate) is the however, we found that the variability at the major storage form of phosphorus in seeds and extraction and reduction phases made the as such forms a variable large component of procedure too imprecise for the determination the diet of many animals and humans.' Phytic of the very small amounts of phosphate acid is a strong chelator ofcations and has been released by the human tissue. shown to cause deficiencies in minerals such as We therefore used a method for phosphate calcium, iron, zinc, and magnesium when measurement using malachite green as the present in excess in the human diet.2 chromogen.15 This improved method does Phytase (E.C.3.1.3.8.) is present in the small not entail phytate extraction before colour intestines of animals such as rats, calves, development. It also has the added advantage chickens, and pigs.3-5 Dietary phytate intake of not requiring protein precipitation as and the ability of the intestine to break deproteination has been shown to release Gastroenterology Unit down phytic acid have been extensively phosphate groups from cellular constituents.15 T H Iqbal studied in animals because of the importance of B T Cooper phytic acid in mineral balance and the poten- and Department of tially deleterious effect ofhigh concentrations of REAGENTS Clinical Chemistry, undigested phosphates in livestock excreta.67 The following reagents were used: sodium Dudley Road Hospital, Despite the fact that humans consume Birmingham large phytate (30 mmol/l), hydrochloric acid amounts K 0 Lewis of phytates, which can cause mineral (5 mol/l); malachite green made up in acidi- deficiencies,8 published works on the ability of fied molybdate (340 mg/l); aqueous TWEEN Correspondence to: Dr B T Cooper, the human intestine to degrade phytic acid are solution (15 ml/l); ammonium molybdate Gastroenterology Unit, surprisingly sparse. There has only been one in hydrochloric acid (34 mmol/l); aqueous Dudley Road Hospital, Birmingham B18 7QH. previous study on phytase activity in the urea solution (360 g/l), aqueous zinc chloride Accepted for publication human small intestine, which found it to be (4 mmol/l); aqueous magnesium chloride 21 December 1993 present but made no mention of the activity of (40 mmol/l). 1234 Iqbal, Lewis, Cooper

0.7 o Buffer 0.6 0.8 *+ 120 mmol/1 phytate Gut: first published as 10.1136/gut.35.9.1233 on 1 September 1994. Downloaded from '-._CO 07 o+ 240 mmol/l phytate -oE 0.5 - 0.6 Vc r -0 0.4 ~0c 0.5_ -0 0.4 .0. 0.3 0.3 0. 0 02 **1 0 0.1 I _ A 0.1 0 1 2 3 4 5 v0 1 2 3 4 5 Phosphate concentration (mmol/1) Phosphate concentration (mmol/1) Figure 1: Working rangefor malachite green method. Figure 2: Effect ofphytate on malachite green methodfor phosphate.

SPECIMENS and at this concentration there is a contribu- Complete human small intestinal specimens tion from undigested phytate to the final were obtained from two previously fit colour. This could be compensated for by subjects during organ harvesting for trans- reagent blanks consisting of buffer with and plantation purposes. One was a 50 year old without phytate. woman who died suddenly from a sub- The concentrations of phosphate in the arachnoid haemorrhage; the other a 40 year incubates and blanks were derived from the old man who died suddenly in a road traffic standard curve. accident. The phytase activity expressed as Rat small intestine was harvested from two ,umolP/min/mg tissue was calculated as: Wistar male rats killed by cervical dislocation. All tissue samples were immediately Phytase activity (p.molP/mg protein/min)= frozen in liquid nitrogen. Subsequently the (Tp-T) - (Bp-B)/(protein concXtime) rat and human donor specimens were kept at -700C. where Tp: tissue+phytate, T: tissue blank, Bp: buffer+phytate, B: reagent blank.

PHYTASE DETERMINATION For the determination of phytase activity the Results brush borders were scraped off underlying tissue with a glass microscope slide before WORKING RANGE OF MALACHITE GREEN homogenisation. METHOD The tissue (approx 200 mg wet weight) was By plotting the absorbance of varying concen- http://gut.bmj.com/ homogenised in 45 ml cold 50 mmol sucrose trations of phosphate standards (Fig 1) plus 2 mmol TRIS HC1 buffer (pH 7.1) with we derived a linear working range of between 20 strokes of a Griffiths tube homogeniser. 1 and 5 mmol/l. The homogenate was centrifuged at 6000 rpm for three minutes and the supernatant separated from the pellet, which was FREEDOM FROM INTERFERENCE BY PHYTATE discarded. An aliquot was taken for alkaline Phosphate standards with added phytate up to on September 27, 2021 by guest. Protected copyright. phosphatase activity, which was estimated on 400 times that used in the final colour reaction the 'Cobas-Bio' automated enzyme analyser product had negligible effect on the using paranitrophenol phosphate as sub- absorbance (Fig 2). strate,16 and for protein determination by the method of Bradford.'7 Homogenate (2 ml) was combined with FREEDOM FROM SPONTANEOUS HYDROLYSIS OF MgCl2 solution (100 p1l) and zinc chloride PHYTATE solution (100 gl). Sodium phytate substrate Reagent blanks comprising buffer with and (100 ,u.l) was then added to the tests and water without phytate showed negligible effects added to the blanks. Reagent blanks consisting on the final absorbance from phytate showing of buffer with or without phytate were that under the experimental conditions included. The tubes were then incubated at there was no spontaneous hydrolysis of phytic 37°C for two hours after which an aliquot acid. (100-500 gl) was taken for determination of inorganic phosphate content. Urea solution (1 ml) was added to this to prevent protein precipitation and then malachite green reagent 0.8- (4 ml) containing acidified molybdate. After 0.7- allowing 10 minutes for colour development, ac c 0.6- the samples were read at 640 nm on a -0 05- Unicam PU86 10 .ii Q Pye U/Visible spectro- Q 04- 0. photometer. 0 For the measurements on rat tissue we 0.31 n.A found that 100 ,u aliquots from the incubation V'Z.0 20 40 60 80 100 120 140 mixture gave good colour formation. The Time (min) much smaller phytase activity in the human Figure 3: Rate ofphosphate liberation during incubation of intestine, however, required 500 gl of incubate rat duodenal homogenate with phytate. Phytase activity in the human and rat small intestine 1235

T1ABLE I Rat duodenal phytase anzd alkaline phosphatase activity anid kinetics. Mean Without Phe (L) (SD) on six replicates shown E 00014 E With Phe 11*0 Cu) 4-

cnEC: Gut: first published as 10.1136/gut.35.9.1233 on 1 September 1994. Downloaded from 0.0012 CD-8 Activitv Vmnax Kmti (ninipiol!7) 0._ CL 0.001 Cl) C)) Phytase (umolP/mg/min) 270 (4.8)X10 X 0 83 4.21 0.0008 C).6 O0 E Alkaline phosphatase (umolP/mg/min) 2.75 (0.02) E CLa Alkaline phosphatase phytase ratio 102 - 0.0006_ 0D.4 m .2 ._ E 0.0002 -I - CL Phytase Alkaline phosphatase -I1ABLE ii Human phytase atnd alkalinle phosphatase. Meani (SD) oni six replicates shown Figure 5: Effect ofphecnv,lalaniole (P/ic) 0o? h2unian1 enzymes. Bars reprcscnt nieanis of three replicates. Phytase Alkaline phosphatasc Ratio (ionolP/mig/olinl.) (uionolP/oig/7niii) (alkalinle pliosphataso.phlx'ase) Duodenum: Subject 1 9 33 (0-8)X 10 4 0-966 (0.07) 1083 HUMAN INTESTINE Subject 2 6.50 (0 19)X 10 4 0.203 (0-01) 363 Jejunum: Subject 1 3.67 (0.5)Xl10 0(614 (0.02) 1702 Human intestinal phytase activity, Subject 2 4 50 (0-12)X10 4 0-647 (0.01) 1526 Ileum: Table II shows the activity of phytase and Subject 1 3-83 (0.4)Xl104 111 (0.02) 2938 alkaline phosphatase at different sites of the Subject 2 3.00 (0-6)X10 4 1 75 (0 01) 6089 human small intestine. It can be seen that there is much less phytase activity in the human small intestine compared with the rat and that this activity diminishes RECOVERY EXPERIMENT lower down the intestine. Known concentrations of phosphate standards Figure 4 shows the distribution of phytase (1-6 mmol/l) were added to rat tissue and alkaline phosphatase activities in the two homogenates in the absence of phytate human intestines investigated. Determination substrate. Recoveries ranged from 90% of the kinetics of human phytase was at a concentration of 1 mmol/l to 83% at beyond the sensitivity of the method because 6 mmol/l. of the low phytase activity found in the human tissue.

RAT DUODENUM Phenylalanine inhibition

Linearity of reaction with time We estimated phytase activity in three http://gut.bmj.com/ Rat homogenate (2 ml) was incubated with human duodenal homogenate replicates with phytic acid for two hours and 100 plI samples and without the addition of phenylalanine were taken every 15 minutes for immediately (50 mmol/l). Alkaline phosphatase was assay of phosphate content. Figure 3 shows the estimated on the same homogenate with and rate of phosphate production obeys first order without the addition of phenylalanine in the kinetics. same concentration. Figure 5 shows that while

the alkaline phosphatase activity was reduced on September 27, 2021 by guest. Protected copyright. by 95% by phenylalanine, there was no effect Phytase activity, Kmi, and Vmax for rat on the phytase activity. duodenum Table 1 shows the results of phytase activity, alkaline phosphatase activity, and kinetics for Discussion phytase. The results are in good agreement This is the first investigation to quantify the with those found by other workers." activity of human small intestinal phytase. In our study the proximal human small intestine was found to contain phytase activity at very low values, which declined distally along the intestine. Human duodenal phytase Plhytase m 2 was 1000 times ] AIkaline phosphatase 2 activity roughly lower than that of alkaline phosphatase and 30 times less C) _ CO)a) than that seen in the rat duodenum. This E 1.5 Xa 07) measured activity did not result from alkaline E _ phosphatase as shown by the lack of inhibition QE of phytase activity under conditions where Cu is almost 05 alkaline phosphatase completely 0~ .E E inhibited. Other workers have found that 0.5 COd phytate itself inhibits alkaline phosphatase. 18 19 We found phytase activity in the rat small intestine comparable with that found by other workers using methods requiring phytate 1\ 11 ^~os e\1 G 0 j\O1Q; extraction. The only previous study on human phytase activity showed the enzyme in Duodenum Jejunum Ileum the human small intestine but did not report Figure 4: Distribution of human intestinal phytase and alkaline phosphatase in two the levels of activity.5 The authors implied that subjects. they saw activity similar to that seen in the rat. 1236 Iqbal, Lewis, Cooper

5 Bitar K, Reinhold JG. Phytase and alkaline phosphatase This is at variance with the much lower activity activities in intestinal mucosa of rat, chicken, calf and found in this study. man. Biochim Biophys Acta 268: 442-52.

1972; Gut: first published as 10.1136/gut.35.9.1233 on 1 September 1994. Downloaded from 6 Pointillart A, Fourdin A, Fontaine N. Importance of cereal Nutritionists have estimated human phytase phytase activity for phytate phosphorus utilization by activity indirectly by studying the breakdown growing pigs fed diets containing triticale or corn. J Nutr in 1987; 117: 907-13. of various foodstuffs by the whole gut 7 Simons PCM, Versteegh HAJ, Jongbloed AW, Kemme PA, normal controls and in patients with Slump P, Bos KD, et al. Improvement of phosphorus availability by microbial phytase in broilers and pigs. BrJ' ileostomies and have suggested that intestinal Nutr 1990; 64: 525-40. phytase activity is very low.920 Our direct 8 Erdman JW. Oilseed phytate. Nutritional implications. J'Am Oil Chem Soc 1979; 56: 736-41. measurements of human intestinal phytase 9 Sandberg AS, Andersson H. Effect of dietary phytase on the activity in vitro would support these findings. digestion of phytate in the stomach and small intestine of intestine to humans. JNutr 1988; 118: 469-73. The ability of the small degrade 10 Davies NT, Flett AA. The similarity between alkaline phytic acid is of considerable nutritional phosphatase and phytase activities in rat intestine and as the mineral their importance in phytate-induced zinc deficiency. importance evidenced by BrJ Nutr 1978; 39: 307-16. deficiencies seen in human populations 11 Rao RK, Ramakrishnan CV. Studies on inositolphosphatase of undegraded in rat small intestine. Enzyme 1985; 33: 205-15. consuming large quantities 12 Varley H. Gowenlock AH, Bell M, eds. Practical clinical phytates.2023 Phytates in the diet are probably biochemistry. 5th ed. Vol 1. London: William Heinemann, in 1980: 883-5. hydrolysed mainly by endogenous phytase 13 Irving GCJ, Cosgrove DJ. Interference by myo-inositol food (for example, yeast phytase) and this hexaphosphate in inorganic orthophosphate determina- breakdown probably occurs largely in the tions. Anal Biochem 1970; 36: 381-8. 14 Cooper JR, Gowing HS. A method for estimating acid environment of the stomach.9 This phosphate in the presence ofphytate and its application to most people diets the determination of phytase. Anal Biochem 1983; 132: would explain why taking 285-7. containing modest amounts of phytates do not 15 Hohenwallner W, Wimmer E. The malachite green in minerals. micromethod for the determination of inorganic become deficient phosphate. Clin Chim Acta 1973; 45: 169-75. An additional recent development in phytate 16 Hausamen TU, Helger R, Rick W, Gross W. Optimal been the hypothesis that conditions for the determination of serum alkaline metabolism has phosphatase by a new kinetic method. Clin Chim Acta phytate may be protective against colonic 1967; 15: 241-5. Phytic acid has recently been 17 Bradford MM. A rapid and sensitive method for the quanti- carcinogenesis.24 tation of microgram quantities of protein utilizing the found to be anti-neoplastic in both mammary principle of protein-dye binding. Anal Biochem 1976; 72: and colon carcinogenesis models in vivo in the 248-51. 18 Martin CJ, Evans WJ. Inactivation of intestinal alkaline rat.25 26 phosphatase by inositol hexaphosphate-Cu(II) coordinate The low level of phytase activity in the complexes. J Inorg Biochem 1991; 42: 161-75. 19 Martin CJ, Evans WJ. Reversible inhibition of intestinal intestine dictates that, from the nutritional alkaline phosphatase by inositol hexaphosphate and its foodstuffs high Cu(II) coordinate complexes. J Inorg Biochem 1991; 42: point of view, comprising 177-84. concentrations of phytic acid should undergo 20 McCance RA, Widdowson EM. Phyates in human nutri-

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