Download Influence of Concomitant Food Intake on tbe Excretion of Orally

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 ..
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