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JOURNAL OF MEDICINAL FOOD J Med Food 9 (1) 2006, 72-76 © Mary Ann Liebert, Ine. and Korean Society of Food Science and Nutrition Influence of Concomitant Food Intake on tbe Excretion of Orally Administered myo- Inositol Hexapbosphate in Humans F. Grases,1 A. Costa-Bauza,1 J. Perelló,1 B. Isem,1 I. Vucenik,2 M. Valiente,3 lA. Muñoz,3 and R.M. Prieto1 1Laboratory oi Renal Lithiasis Research, Universitary lnstitut oi Health Sciences Research, University oi Balearic Islands, Palma oi Mallorca; 3Deparlment oi Chemistry, Aulonomous University oi Barcelona, Barcelona, Spain; and 2Deparlment oi Medical and Research Technology, University oi Maryland School of Medicine, Baltimore, Maryland ABSTRACT myo-Inositol hexaphosphate (lnsP6) widely occurs in plant seeds. At prdsent, some important benetits of InsP6 I for human health have been described. The purpose of this study was to find the best condition for the optimum absorption of ora11y administered InsP6, evaluated by InsP6 urinary excretion. The influence of different stomach conditions (empty, empty with an alkalinizing agent, and full stomach) on the effects of oral administration of InsP6 and its urinary excretion was investigated in six healthy subjects on an InsP6-poor diet, given 400 mg of calciumlmagnesium salt of InsP6 as a single dose. The basal urinary excretion of InsP6 on an InsP6-poor diet (50.91 :t 15.09 /Lg) was significantly lower than that found when an InsP6-normal diet was consumed (100.09 :t 26.42 /Lg) (P < .05). No differences were observed in the areas under the curve of accumulated excretion at 8 hours among the three different stomach conditions studied, suggesting that the overa11InsP 6 absorption took place independentIy of the stomach state (full or fasted) and indicating that the InsP6 absorption also takes place during the intestinal transit. Thus, if InsP6 supplements of vegetal origin are consumed to maintain the optimum [nsP6 levels needed for a healthy status, tbese supplements can be consumed either during or between meals with the same efficacy. KEY WORDS: • excretion • rnyo-inositol hexaphosphate • phytate INTRODUCTION 60-80% of the phosphorus present in such organs may be InsP 6. The content ofInsP 6 found in seed plants ranges from 0.4% to 6.4%. Consequently, this substance is ahuman dietary component. 1.2 Moreover, InsP 6 is also found in all mammalian tissues and biological fluids, although at notably lower amoun15 than in seeds; its concentration in plasma is 0.26 :t: 0.03 mg/L,3 and in urine 2.94 :!:: 0.20 mg/L.4 On the other hand, these detected levels are dearly related to the dietary ingestion of InsP6; consumption of an InsP6-poor diet significantly decreases the urinary and plasma levels of InsP6.4.5 At present some important benefits of InsP6 for human health have been described. Its capacity as a crystallization inhibitor, important in the prevention of pathological calcifications such as renal calculi6-1O or ectopic calcifications, has been demonstrated. Furthermore, the antioxidant capacity12-14 of InsP6 and its protective action against cancerl5-18 have been also shown. m YO-INOSITOL HEXAPHOSPHATE (InsP6 or phytate) widely occurs in plant seeds and/or grains mainly in cereals, legumes (beans), and nuts, but also in roo15, tubers, fruits, and vegetables, although in much smaller quantities. InsP6 is contained primarily as a salt of mono- and divalent cations (Mg2+, K+, Ca2+, 2n2+, Fe2+, and Cu2+) in discrete regions of grains and seeds.1 The accumulation site of phytate in grains and seeds is within the subcellular single-membrane partic1es, aleurone grains, or protein bodies as globoids. The aleurone grains are located in the aleurone cells of monocotyledonous seeds such as cereals.2 In dicotyledonous seeds such as legumes and many other seeds, globoids are located within the proteinaceous matrix of protein bodies. Globoids are present in the cotyledons of dicotyledonous seeds but not in their seed coats. As much as Manuscript II InsP6 is a heat-stable compound. However, food processing either at home or in industry-including a variety of methods, such as soaking,cooking, germinating, seed irradiating, extruding, milling, frying, fermenting, roasting, microwaving, and severalcombinations of these methods- received 1 October 2004. Revision accepted 27 December 2004. Address reprinl requesls lo: Prof Dr. F. Grases, Laboralory 01Renal Lilhiasis Research, Universilary 07122-Palma Inslitul of Health Sciences Research, University de Mallorca, Spain, E-mail: [email protected] 01 Balearic • stomach conditions Islands, 72 CONCOMITANT FOOD INTAKE Wlrn TABLE 1. DEMOGRAPHICS OF HEALTHY 73 linizing agent, and full stomach) on the oral administration, absorption, and subsequent urinary excretion of InsP6. SUB)ECTS SELECTEO FOR STUOY Characteristic ORAL INSP6 Number Number oí volunteers MA TERIALS AND METHODS 6 Study design and sa1;nplecollection Sex 3 3 Male Female 41 Age (years) Weight (kg) 65 :t 5 :t 4 :t standard error: Data are expressed as mean may decrease the InsP6 content of foods. This decrease depends upon the food to be processed and upon' the type of processing method employed. Stilllittle is known about the turnover of InsP6 in the organism. It has been demonstrated that after InsP6 depletion due to its elimination from diet, InsP6given as a supplement was quickly absorbed, reaching the maximum concentrations in plasma 4 hours after the ingestion.3 However, these maximum levels indicate an overall low percentage of absorption. It seems that there is an optimum amount of InsP6 absorbed, above which no increase in the excreted amount can be achieved. It seems that the excreted amount of InsP6 is not affected by the type of InsP6 salt used, since there was no difference in excretion when InsP6 was given either as sodium or calcium/magnesium, or either pure or as a plant component. 5,7 To better understand the factors implied in the observed InsP6 urinary excretion, which are related to InsP6 absorption, we conducted a study investigating the effects of three different stomach conditions (empty, empty with an aIka- Free-Diet t InsP6-Poor Diet •• Ifu~"'**~~Iii'~~\1í1k'ii!!Iiíii\iI..~!'¡"tii~~L'i!;I!~1It\\!!W!~~ Urine sample 150ml Tap water 150ml water + : Time ..•• Id.Tapsample Breakfast 21phytln V 150ml Tap water 11•• Urine 462 Fasting 18 ;, + 150ml water Fastlng Fasting •• Urine Urlne 8-2 •• Urine sample o 15 400mg 2~::-2 W Urine sample +Tap O sample ••.+l,OOOmg Sodium ·i,. + 150ml + 150ml Tap water Carbonlc water 4" ;:)"Id. ¡_ ,e Six healthy subjects (Table 1) were selected to study the effect of a single oral dose of InsP6 on its urinary excretion, as a function of three stomach conditions: (1) empty, (2) empty with an alkalinizing agent, and (3) full stomach. The basal level of InsP6 urinary excretion was determined before experiment initiation in subjects following a non-lnsP6restricted diet. For this, after fasting, ovemight, early in the moming (7:00 hours) they voided the urine accumulated in the bladder ovemight and only drank 150 mL of tap water. Two hours later, a urine sample was collected to determine the InsP6 content The complete experiment was conducted in three phases. In the first phase, all subjects were subjected to an InsP6poor diet for 15 days (all types of cereals or cereal-based foods, legumes, beans or bean-based foods, 'nuts, nut-based foods, and other vegetable seeds were totally exduded; all other foods USedhad a content of less than 0.1 % phytateI). On day 15, after ovemight fasting, early in the moming (7:00 hours) the subjects voided the urine accumulated ovemight and only drank 150 mL of tap water. Two hours later, the frrst urine sample was collected (basal2-hoururine sample), and the subjects immediately ingested 400 mg of calcium/magnesium phytate (phytin of Triticum aestivum, supplied by Authex Laboratorios, Marratxí, Mallorca, Spain) with 150 mL of tap water. Four new urine samples were collected after 2, 4, 6, and 8 hours, and after each collection of urine, 150 mL ofwater was drunk (the last sample was col- 8 t 6 Fasting Voided bladder bladder 400m, ,h~," O 1" Id. }~ 400nng phytin Urine sample + 150o.n., .",,, FIG. 1. Overview of the experimental procedure. Id, previous step repeated. 74 GRASES ET AL. TABLE 2. INSP6 URINARY EXCRETION IN A BASAL 2-HoUR all scheme of the study design is shown in Figure 1. The protocol of tbis study was approved by the Ethics Committee ofthe University ofBalearic Islands, Palma de Mallorca, Spain. An informed consent was obtained from all subjects before study. URINE SAMPLE IN EXPERIMENTAL SUBJECTS FOLLOWING A FREE DIEr OR AN INSP6-POOR DIEr Diet Free (n = 6) InsP6-poor (n InsPó = 100.09 50.91 6) (¡.Lg) ± ± 26.42 1S.09a InsP6 detennination Data are expressed as mean ± standard error. Student's t test was used to determine statistical significance between means. ap < .05 versus free diet. Fresh urine was acidified with HCI (1: 1) until pH 3-4. An aliquot of 5.0 mL of urine was transferred to a column (inner diameter 4 mm) containing 0.2 g of anion exchange resin (AG 1 X 8, 200-400 mesh, Bio-Rad, Hercules, CA). The first eluate was discarded, and the column was washed with SO mL of 50 mM HCl. This second eluate was also discarded. Then, the column was washed with 3 mL of 2 M HN03• The determination of phytate was carried out through direct phosphoros analysis of this last eluateby inductively lected at 17:00 hours). Then, the subjects followed the InsP6poor diet. After 3 days had elapsed as a stabilization period, the volunteers repeated the same procedure as described for day 15, but this time instead of tap water, they drank carbonic water (Vichy Catalan, Barcelona, Spain) with a chemical composition of 2,212 mg/L bicarbonate, 610 mg/L ch1oride, 47.5 mglL sulfate, 7.3 mg/L fIuoride, 1,137 mg/L sodium, 50.1 mg/L potassium, and 1.4 mg/L lithium. They ingested 400 mg of calcium/magnesium phytate with 1,000 mg of sodium bicarbonate with 150 mL of carbonic water. After 3 days had elapsed as a new stabilization period, the subjects repeated the same procedure as described for day 15, but at tbis time tbey ingested 400 mg of calciumlmagnesium phytate, togetber witb an InsP6-free breakfast, consisting of two fried eggs witb bacon, one yogurt, or tbe same quantity offresh cheese with 150 mL oftap water. The over- coupled plasma atpmic emission spectrometry using the corresponding calibration curve. All chemicals used were of analytical-reagent grade. A Model 2000 inductively coupled plasma atomic emission spectrophotometer (Perkin Elmer, NorwaIk, CI) was used for total phosphorus determination. Statistical analysis Area under tbe accumulated excretion curve (AUC) expressed as JLg • hour were calculated for each subject by tbe trapezoidal method. Values in tbe figures and tables are ex- 600 ....-Empty .-ll-Empty with an alkalinizer ! e o iu •... -o- FuI!stomach 500 )( W .~ 400 ns ¡:e ::::1 -.!! -g 300 :1 E u~ 200 o( Di ..:!- 100 ••• A. '" e 0_ O 2 3 4 5 6 7 8 Time (hours) FIG.2. InsPó accumulated urinary excretion during an 8-hour period after ingestion of a single dose of 400 mg of InsPó as a phytin of T. aestivum following an InsP6-poor diet in the three stomach conditions. Data are expressed as mean ± standard error of six experimental subjects. CONCOMITANT pressed as mean :t standard error. One-way analysis ofvariance was used to calculate significance of differences among the three groups. Student's t test was used to assess differences of means. Conventional Windows programs were used for statistical computations. A probability of P < .05 was used to assess statistical significance. RESULTS The basallevels ofInsP6 in urine of subjects on an InsP6poor diet were significantly lower (50.91 :t 15.09 ¡Lg) than those found when an InsP6-normal diet was consumed , (100.09 :t 26.42 ¡Lg) (P < .05) (Table 2). The accumulated excretion curves of InsP 6 for each of the different stomach conditions (consuming an' InsP6-poor diet) after ingestion of a single InsP6 dose (400 mg of calciumlmagnesium salt) , as a function of time during the 8hour periad are shown in Figure 2. No differences in the A UC of InsP 6 accumulated excretion were found among the three different stomach conditions studied (empty, empty with an alkalinizing agent, and full stomach) (Table 3). This indicates that the different stomach conditions contributed to the InsP6 absorption The percentages of were not significantly conditions studied, as in approximately the same amounts. oral dose urinary excretion of InsP6 different among the three stomach shown in Table 4. DISCUSSION To understand the beneficial activity of InsP 6 and to proper1y design clinical trials for its use, it is very important to elucidate the absorption, metabolism, tissue distribution, and excretion of InsP6. InsP6 contents in organ and fluids depend on its dietary intake3.4; the levels found in rats fed with a diet without InsP6 were around 100-fold less than those found in rats fed with an InsP6-rich diet. In celllines, treatment with extracellular InsP6 increased the intracellular myo-inositol trisphosphate levels, but did not modify the InsP 6 intracellular contents.19 The InsP 6 intracellular leve1s I in celllines were of the same order of magnitude as those TABLE 3. AUC AN 8-HoUR OF ACCUMULATED URINARY EXCRETION DuRING PERIOD AFTER INGESTION OF 400 MG OF CALCIUM/MAGNESIUM PHYTATE IN EXPERllvlENTAL SUBJECTS FOLLOWING AN INsP6-PooR CONDmoNs: DlET COMPARING THREE STOMACH EtvlPTY, EtvlPTY WITH AN ALKALlNIZER, AND FULL Stomach condition Empty Empty with an alkalinizer Ful! AUC 75 FOOD INTAKE WITH ORAL INSP6 (JLg 1,209 1,198 1,117 ± ± ± hour) 183 96 123 Data are expressed as mean ± standard error of six experimental subjects. One-way analysis of variance was used to calculate the significance of differences among three groups. No differences were observed. TABLE 4. PERCENTAGE OF AN ÚRAL DosE OF INsP6 EXCRETED THROUGH URINE DuRING AN 8-HoUR PERIOD Stomach condition Empty Empty with an alkalinizing agent Full InsP6 (% oi oral dnse) . 0.39 0.39 0.38 ± 0.02 ± 0.03 ± 0.04 Data are expressed as mean ± standard error of six experimental subjects. One-way analysis of variance was used to calculate the significance of differences among the three groups. No differences were observed. found in tissues of animals fed with a diet without Insp6.19 All these data suggest that the increase in InsP6 tissue levels must be attributed to the increase of the extracellular InsP6 (plasma and interstitial fluid). These results also demonstrate that the majority of InsP6 present in the organs and tissues is of dietary origin, and not a result of endogenous synthesis of InsP 6. Thus, InsP 6 must be supplied by foad and/or supplements to maintain adequate levels in different organs and tissues. Moreover, it was found that the urinary levels were directly related to the plasma values, and, therefore, InsP 6 in urine constitutes a marker of InsP 6 deficiency in the organism.3 The present study was conducted to evaluate the bioavailability of InsP6 when taken on a full versus an empty stomach with or without an aIkalinizing agent. The results obtained indicated that the profIle of InsP6 urinary excretion (Fig. 2) and the AUC of accumulated excretion curve (Table 3) at 8 hours were the same for the three different stomach conditions studied. If the gastrointestinal InsP6 absorption would be preferentia11y in the stomach, the absorption would take place mostly through the stomach walls and would require InsP6 mainly to be in its neutral protonated formo Consequently this would be more favorable on an empty stomach, with a low pH value, and would be independent of the ingested salt. In fact, previous studies have demonstrated that there is a maximum excretion level that cannot be exceeded by ingesting higher amounts of InsP6.3 In that study InsP6 was given with different formulations, but the excreted amounts of InsP6 were not affected by the type of InsP6 salt used, either calciumlmagnesium or sodium, with empty stomach conditions.3 The results obtained here suggest that the overall InsP6 excretion takes place independently of the stomach conditions, indicating that InsP6 absorption also happens during intestinal transit, independent of whether specific tansporters exist or not. The percentage of the oral dose excreted through urine during an 8-hour period is very low (around 0.4%). This indicates that absorption rate of InsP6 is low, the same as has been previously observed with other synthetic phosphatecontaining molecules such as diphosphonates.20 In conc1usion, the absorption and urinary excretion of InsP6 are independent of the stomach status, suggesting that if InsP6 supplements are consumed to maintain optimum 76 GRASES ET AL. InsP6 status, these can be taken either during or between meals with the same efficacy. ACKNOWLEDGMENTS One of the authors (IP.) expresses his appreciation to the Spanish Ministry of Education, Culture and Sport for a fellowship in the FPU programo AIso, B.1. expresses his appreciation to the Conselleria d'Innovació i Energia del Govern de les mes Balears for a fellowship. Ibis work was supported by the Conselleria d'Innovació i Energia del Govern de les Illes Balears (grant PROIB-2002GCI-04) and by project BQU 2003-01659 of the Spanish Ministry of Science and Technology .. REFERENCES 1. Reddy NR: Occurrence, distribution, content and dietary intake of phytate. In: Foods Phytate (Reddy NR, Sathe SK, eds.), CRC Press, Boca Raton, FL, 2002, pp. 36-63. 2. Ogawa M, Tanaka K, Kasai Z: Isolation of high-phytin-containing particles from rice grains using an aqueous polymer two phase system. Agric Biol Chem 1975;39:695-700. 3. Orases F, Simonet BM, Vucenik 1, Prieto RM, Costa-Bauza A, March JG, Shamsuddin AM: Absorption and excretion of orally administered inositol hexaphosphate (IP6 or phytate) in humans. BioFactors 2001 ;15:53-61. 4. Orases F, Simonet BM, March JG, Prieto RM: Inositol hexakisphosphate in urine: the relationship between oral intake and urinary excretion. BJU Int 2000;85:138-142. 5. Grases F, Simonet BM, Prieto RM, March JG: Phytate levels in di verse rat tissues: influence of dietary phytate. Br J Nutr 2001;86:225-231. 6. 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