Studies in Calcium Metabolism: Effect of Food Phytates on Calcium-45 Uptake in Children on Low-Calcium Breakfasts (Journal of Nutrition Vol. 54 No. 4 10 December 1954)

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STUDIES I N CALCIUM METABOLISSI: EFFECT OF FOOD PHYTATES ON CALCICV4j UPTAKE Department o f Physics, Uassarkusc.tf~I xs fit ute of lech nd ngy, Cambridge Department o f Senropaychiatry, Hurrard Medical Srhwl, Rortor, aid 5 (Received for publicatioo June 4, 19M) Although many investigators have studied the influence of the uptake of calcium by human beings, the mechanism of the phytate effect is not well understood. This is partly due to the metabolic balance technique used hr these workers I the inosi to1 hexaphosphates (commonly called phytates) oni i 'Preaented a t the Annual Meeting of the American Institute of Sutrition, Chicago, Illinois, April 6-10, 1953. 'Contribution No. 205 from the Department of Food Technolog?, JIaMaehusetta Inititute of Technology. 'Partial aupport to thia study w i s provided bp a grant of the Quaker Oats Companp to the Department of Food Technology and hp Contract IT(30-1)-953 of the Atomic Energy Commission with the Department of Physics. 'The data in thin publication are taken from the thesis (1953) pmsented by Felix Rronner t o tbe Department of Fond Technology, Massnrhusetta Institute of Technologp, in partial fulfillment of the requirements for the degree of Doctor ' Authorization for the we of restricted dowa of radiocalcium (Ca") in patients inatitutionnlized for mental inadequacy was granted throuph the Subcommittee on Human Applicationa by the Isotope Division of the Atomic E n e r m Commission. The radiocalcium was obtained from the Oak Ridge S ation d Labor a to ~ . Quaker Oats Fellow, 1950-1951. Present address: The Rockefeller Institute for Medical Reaeareh, S e w York 01, S. Y.

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524 FELIS BROS SEB ASD OTHEBS Walker et al., ’48; Sicolaysen and Xjaa, 51), with all the difficulties inherent in the interpretation of results (Hegsted, ’52). A new approach to this problem became possible when radioisotopes %-ere made available for biochemical research. Isotopes were used by Sharpe et al. (’50) in a study of the effect of phytates on the absorption of iron in human subjects. In the investigation reported here, radioactive calcium (Ca‘j, half-life 180 days ’) was used to study the effect of phytates on the uptake and escretion of calcium in a similar group of adolescent boys. As in the earlier study (Sharpe et al., ‘$0) use was made of the “one meal technique,” in which the isotope is administered in a single test meal. Since the mass effect of the isotope is negligible, no adjustment period is needed and the study can be terminated as soon as the effects under investigation have been observed. This technique has the real advantage of permitting conditions which are quite similar to those prevailing under normal living, and thus largely circumvents the influence of seasonal factors (NcCance and Widdowson, ’43). On the other hand, the technique cannot be used to study the adjustment on the part of the body to unusual conditions of intake, for it tends to dramatize effects which over long periods of time may be of minor significance. In the present investigation the effect of a phytate-rich cereal (oatmeal) on the uptake of calcium was compared with in conjunction with cooked breakfast cereals, was used as the principal source of calcium. The effect of food phytates was further compared with :hat of soluble sodium phptate which was added to farina. In order to neutralize any effect due to the solids content of the food (Sharpe et al., ’a),all be 180 d r y . Shortly aftrr the exprimental pnrt of this inrestipntion was polleluded, Delrnr. and Poolc (‘3)puidishrd a half.lifr \ n h c of 163.5 2 4 day8 (maximum error). Because of the usc of an rxpriiiiriital conversion factor, LI discussed in thr wetion of Analytical Methods. the dntn presented here do not depend on knotvledge of the hdf.life of the iaotop.

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PHTTATES AS0 CA" UPTAKE IS CHILD RES 525 re do not meals were equalized to a common solids content by the addition of farina. The general procedure was to mix the radiocalcium intimately with the milk of the test meal and then to measure the calcium and radiocalcium content of the serum, urine and feces at interrals following ingestion of the test meal of lvhicli the milk was a part. .\ criss-cross design was used, that is, all individuals were given two tvst nicals tlircc weeks apart, to permit both paired niid group coniparisoiis. -1 three-week interval was chosen take of 0.8.5 pc ('a'.-' thc I c w l of radiocalciuni in blood, urine and fcw sivas iicg1igil)lc ilftt'r that time. This finding wax Sineteen adolescent boFs, of inadequate intelligence but otlierwise nornial, who were institutionalized in a state scliool under uniform nutritional and environmental conditions, volunteered for these esperiments (table 1). The subjects were preparcd for the stud? 1):- ,giving each a daily supplement of one quart of inilk nnd one multivitaniin talilet I( durinq oiic month prior to tlie start of the experiment, and until i t with pliytic phosphorus naturally present in the oatmeal: a farin;i lwcakfast (nical 11) which contained no phytatte: and The conipo4tion of these meals is showii in tul)le 2. RadioCa), wtis added to tlie milk which was then mixed intimately into the cereal of each meaL

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I PHYTATES ASD CA'' UPTAKE IN CEILDREN 529 (freshly prepared with redistilled H,O) after the first and second centrifugations. After the third centrifugation, the oxalate was dissolved in 2 s perchloric acid (HtlO,) and Each sample w a s analyzed in triplicate. of triplicate analyses \\-as usually 3% or better. Analyses were repeated when the precision was not satisfactory. Recovery of nddcd calcium in a series of serum saiiipIc* !IT';.: and in i l sci-ies of urine Feces eaniples were prepilrcd by niakiiy a slurry of feces with toluene and water until a creamy, hoinoqeneous emulsion was obtained. The slurry \\-as then sampled into moisture dishes and crucibles. The moisture determinations served as clwcks 011 the homogeneity of the slurry and as a basis of comparison when an ashing had to bc repeated. The slurry in the crucibles was dried orenlight, charrcd over a Bunsen burner, dry-ashed owrnight in a m d e furnace ( 3M°C.) and wet-ashed with ti mixture (1: 1) of conc. HSO, and solution was analyzed for calcium content I)? precipitation of the calcium a s the osdnte and titration with perchloratoceric acid. The coefficient of variation of duplicate feces samples. each analyzed in triplicate, generally ranged from 3 to 5 % . The recovery of added c;ilciuni in test experiments d ucrlysis for rod iocdrirr iii, Rndiocalcium analyses were carried out by a modification of mcthods oriKiiiall>- developed at the Radioactivity Center, Massachusetts Institute of TechScrrcrir. The calciuni of 5 ml serum saniplw was precipitated directly as the oxalate and isolated hy the centrifugation procedure given above. The wash volutne. however. wa!: The coefficient of variation

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530 FELIX BBONNEB AND OTHEBS 3 ml and the supernatant fluid was removed by a hooked apillarp (Assoc. Official Agricultural Chemists, ’46), in order to avoid losses that might occur when the larger ( W m l ) tubs are inverted. After the oxalate was dissolved in 2N Hclo,, calcium chloride was added as a carrier in amounts s&icient to bring the total calcium to 4.0mg; then the calcium was reprecipitated. After standing overnight, the solution was poured through a two-section sintered glass filter apparatus, and the calcium oxalate was collected on a 24 mm disc of filter paper (Schleicher and Schuell, NO. 5i6). The filter paper was glued on a copper planchet and the calcium45content of the sample determined by counting in an automatic apparatus (1.2 mg/’cm2) counter. Suitable Caa3 standards which had been prepared under identical conditions were emploped in all counting runs. I n addition, electroplated Co60 standards Irvine, Jr., unpublished). Samples were counted in three cycles, to an accuniulation of 30i2 counts. [-rim? samples were prepared by direct precipitation. The precipitated calcium osalate was removed by filtration (Schleicher and Schuell, red ribbon paper) when more than 25ml of urine mere required to prepare a sample containing 4mg of calcium or slightly less : otherwise centrifugation was employed. The procedure used for preparing and counting planchets of urine samples \vas identical with that emploped with the serum samples. -\\-hell ~ic~cc~sary,a carrier was added to bring the calciuiii content to 4.0mp. \\’hen urine samples were not clear, the urine was ashecl by a procedure similar to that used for the feces samples, and the resulting ash solution was tlicii i i ~ c di n tlic Iwcparntioii of samples to be .jstlcd solutions of the ,/‘cw.q were ali illy zd for radiocalcium content by pipettins out a qmntity containing 4 m g of calto bring the cnlciuiii coiitcnt to 4.0 nig. The calcium was then precipitatd with nnmonium osnlatc and the procedure from

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analysis the procedure outlined by Snedecor ('46) for nonorthogonal data was followed. Sertrni. There was an appreciable difference in the serum specific activitr of the three groups: the highest level was observed in the individuals on the farina breakfast, while those \\-I10 had received the oatmeal eshibitcd only 60% of the specific activitr level of the farina group. The individuals \vho had received the sodium phptate had the lowest radiocalciuni l w i ~ l ,4T)T that of the farina group. Statistical analysis (tal)lc 4) revealed that thcse treatment effects were extremely of variaiice indicated that the esperiments differed from each observed in cspcrinieiit B would lead one to expect this. However, this does riot detract from the significance of the treatment proper, particularly since the F-value associated with the trcatmcnt cffcct is very nearly 11 times larger than that associated with the difference hetiwen esperiments. [-rittt>.''' It is to lw espcctcd that the 3pccific activity of the urine ivoulc! l ~ b3iiiiilar t o that of the seruiii. The specific activity \\-as lii~licsti n tiic urine o f the group on farina breakfast, was lowezt in the ui*iiic cscreted hy thc group which

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?e in the serum Thest lerel was ered from each to expect this. ies larger than activity of the tan t diff ercnce I of tlie first thrw

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in the endogenous calcium output would not have been expected under the experimental conditions. Gnpublished results with intravenously injected ~ alc i urn ' ~indicate that less than 570 of the calcium absorbed from the intestinal tract is lost in the feces within 5 days. This loss is less than the deviation of the results reported in tables 4 and 5. dremge per cent dose of Cay crcreted i n feces Excluding subjects 37 and 42 in experiment A. 111-Farina plus phytate breakfast. The agreement between the data on the feces and serum samples shown in table i is of interest. Data on serum and feces samples I ? were obtained independently, and pet the ratios of the mean specific activit?: of the serum data are the same as the inrerse ratios of the feces data. This may be taken as evidence that, under the conditions of this experiment, the fecal cnlciuni'~ was esscntially unabsorbed cal- 'I .it prcacnt, it is not pcrniissiblt~ t o esrlude the possilbility that calcium exchange takes place hctwren the iiitcstinnl tract and the plnsuia (Sorrh, '52). tent administered to the eqwritrwntnl groups wrrr the S:IIIIC..the esperimentrl effect, whatever its n:iture, in all lik(~lili*iodwas indurrd by the phytnte molecule. The assuniption that :ill of the c:iIciiiin& riot reeorerrd in the feres reprerentc calcium uptake merely ni:iyiiitirs tlir alisiilute pliTtate effczt : its relntke effect in all probalditr woitlil lw uurlinnped. wliaterer tlie true uptake.

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leave little room for doubt that phytate does interfere with calcium uptake, and that less calcium enters the body when phytate-containing cereals are eaten than when phytate-free cereals are ingested. On the other hand, the absolute quantity of calcium rendered unavailable as a result of the action of the food phytate was SO small (15 mg) that phytates cannot be very significant in the usual G. S. diets. This will be discussed in greater detail elsewhere (Bronner, Harris, Maletskos and Benda, to 1. Sineteen adolcscent boy*, of inadequate intelligence, but otherwise normal, who were institutionalized under uniform nutritional and other environmental conditions, were giren three types of breakfast low in calcium: (I) oatmeal, (11) farina, and (111) farina and sodium phytate. These meals contained, respectively: 55, 60, and 60ml of milk: 91, 83, and 83mg of calcium; 116, 0, and i 8 m g of phytic phosphorus, and 0.83, 0.85, and 0.85 pc of radioactive calcium (Ca';)). Calcium uptake was studied by measuring the content of seruni, urine and feces for a period of 5 days. Individual variations were controlled by placing each subject on two different C'n45-lnbeled breakfasts, 30 days 2. The uptake of ('3': h? the boys on the oatmeal breakfast was i4P as great as that of the boys 011 the farina breakfast. Similarly, the uptake of Cn4j by the boys on the farina plus phytate meal \vas 45% that of farina meal. These differences proved to be statistically significant on a 5% proba3. Si_qnificantly less Ca4j was taken up in the presence of sodium phytate than in thc presence of an equivalent quantity of phytic phosphorus supplied by oats.

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PHSTATES AND CA" UPTAKE IN CHILDREX 541 The technical assistance of blrs. Alice J. Liu Chen and lliss Lee H. Weiss is gratefully acknowledged. Thanks are due to Dr. Malcolm J. Farrell, Superintendent, and the staff and personnel of the Walter E. Fernald State School, for assistance in carrying out the research. Thanks are also due to the boys themselves for their willing cooperation. BROSNEB,F. 19.52 P1i.D. Thesis, Massachusetts Tlistitllte of Tcchnologc. CLARK, E. P.. .\SD .T. 13. COLLIP 1925 Detcrniinntion n f Ijlood scruin calciiiiii. of iron and phospliorus in ferric phrtnte. Ind. Eng. Chem. Anal. Ed., The stabilitc of complexes between calcium and orthophosphnte, polymeric phosphate, and phptate. Arch. HAFIN,P. F. 1945 Radioact ire iron. Procedures, purification, electroplating and nnalrsis. Ind. Eng. Chem. -4nal. Ed., 27: 45. HEGSTED,D. If. 1952 False estimates of adult requiremrnts. Sutrition Rev.. HOFF-JORGFSSES,E. 1944 Inrestipations on the solubilitc of calcium phytate. Det. kgl. Danske Yidcnok. Selsk. Mat-Fps. aleddel., if, Xo. 7. KREBS,11. .\., ASD ti. >IF.LLASBT 1943 The effect of national rheatmeal on the ahsorption of calcium. Biochrm. 6..37: 466. Lon~r,0.€I., .\SD .T. A. LOPEZ The determination of i a or p nie phosphate in the presence of labile phosphate ester% J. Biol. Chem, 26?: 421. adults on white and brown bread dietaries. J. Phgsiol., 202: 44. 194211 Mineral metabolism on dephptinized bread. Ibid., 101 .- 304. 1943 Seasonal and annual changes i n the calcium metabolism of 1949 Phytic acid. Brit. J. Sutrition, 2: 401. Methods of Analysis. Sixth Edition. aktiren Calcium. Erperientia, 8: 71. CHRISTESSES 1946 On phptic acid. its importance in metaholism a n d its rnzTmatic clrarage in bread supplemcntrd with calcium. Bio

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STUDIES IX CALCIUM METABOLISM. EFFECT OF FOOD PHYTATES OX CALCIUM45 UPTAKE IN BOYS Oh' A MODERATE FELIX BBONNER. AND ROBERT s. HARRIS Department of Food Technology Deportment of Phlyricr, Yauachwettr …