Aleurone Flour Is a Rich Source of Bioavailable Folate in Humans

Abstract

With the use of novel milling technology, it has become commercially viable to isolate the aleurone layer of cells from wheat grain and to prepare a novel flour from this fraction that has a natural folate concentration of approximately 500 microgram/100 g. The aim of this study was to determine the relative bioavailability of natural folate from aleurone flour when ingested as a cereal. Using a series of randomized, short-term intervention trials with a cross-over involving eight men and eight women aged between 29 and 50 y, we compared the increment of plasma folate following ingestion of 1) 100 g wheat bran cereal (low folate control), 2) 100 g aleurone cereal, and 3) a tablet containing 500 microgram folic acid taken together with 100 g wheat bran cereal (high folate control). Folate absorption was measured by estimating the area under the plasma folate concentration versus time curve. The extent of increase in plasma folate over the 7-hour period following ingestion of aleurone cereal was more than fourfold greater than that observed following the wheat bran cereal (P < 0.0001) and not different from that observed following the 500 microgram folic acid tablet taken with wheat bran cereal. Differences were significant when data for males and females were analyzed separately (P < 0.001). This study has shown that cereal made from wheat aleurone flour is a good source of bioavailable, natural folate.

Full text

Human Nutrition and Metabolism
Aleurone Flour Is a Rich Source of Bioavailable Folate in Humans
1
Michael Fenech,
2
Manny Noakes, Peter Clifton and David Topping
Commonwealth Scientific and Industrial Research Organisation, Human Nutrition, Adelaide, SA, Australia
5000
ABSTRACT With the use of novel milling technology, it has become commercially viable to isolate the aleurone
layer of cells from wheat grain and to prepare a novel flour from this fraction that has a natural folate concentration
of ;500
m
g/100 g. The aim of this study was to determine the relative bioavailability of natural folate from aleurone
flour when ingested as a cereal. Using a series of randomized, short-term intervention trials with a cross-over
involving eight men and eight women aged between 29 and 50 y, we compared the increment of plasma folate
following ingestion of 1) 100 g wheat bran cereal (low folate control), 2) 100 g aleurone cereal, and 3) a tablet
containing 500
m
g folic acid taken together with 100 g wheat bran cereal (high folate control). Folate absorption was
measured by estimating the area under the plasma folate concentration versus time curve. The extent of increase
in plasma folate over the 7-hour period following ingestion of aleurone cereal was more than fourfold greater than
that observed following the wheat bran cereal (P , 0.0001) and not different from that observed following the 500
m
g folic acid tablet taken with wheat bran cereal. Differences were significant when data for males and females
were analyzed separately (P , 0.001). This study has shown that cereal made from wheat aleurone flour is a good
source of bioavailable, natural folate. J. Nutr. 129: 1114–1119, 1999.
KEY WORDS:
●
aleurone flour
●
folic acid
●
bioavailability
●
wheat bran
●
humans
Wheat aleurone flour is a novel food product that has the
potential to make an important contribution to the intake of
natural folate. The aleurone cells, together with the germ,
contain the wheat grain’s essential nutrients required for the
growth and development of the embryo (Clysedale 1994,
Saxelby and Venn-Brown 1980). The phytochemicals, vita-
mins, and minerals in aleurone cells may be lost when wheat
grain is refined. A unique and commercially viable milling
process that enables the isolation of the aleurone cell layer and
at the same time splits the cell walls to release the contents of
these cells has recently been developed (by Goodman Fielder
Pty. Ltd., Australia) (Stenvert 1995 and 1997). A schematic
representation of the isolation of aleurone is shown in Fig. 1.
The sheared aleurone cells together with a small amount of
wheat germ was formulated into a novel aleurone flour
(ALF)
3
. The aleurone flour described in this study was avail-
able commercially in Australia and internationally for more
than 12 months and is sold widely as a major ingredient of
bread and pasta. One of the most notable features of the
composition of this product is the high level of folate that is
present, a concentration between 400 and 600
m
g per 100 g
wet ALF. This natural level of folate is higher than that
observed in wheat bran, fruits, and vegetables (usually between
20 and 200
m
g/100 g wet) (Bailey 1995, Subar et al. 1989) and
is comparable to folate/folic acid levels in fortified flour and
cereal that provide 50% Recommended Dietary Intake (RDI)
per serve (assuming an RDI of 400
m
g and a serving size of
40 g wet weight) (Crane et al. 1995).
Folate is now recognized to play an important role in the
prevention of neural tube defects in the fetus (Czeizel and
Dudas 1992, Medical Research Council Vitamin Study Re-
search Group 1991). There is also increasing evidence that an
above average intake of folate may help reduce plasma homo-
cysteine, a risk factor for cardiovascular disease (Boushey et al.
1995, Kang et al. 1992), and DNA damage, a risk factor for
cancer (Blount et al. 1997, Fenech 1996, Ma et al. 1997).
There is some concern that eating foods that are naturally rich
in folate may not provide for a large enough and reliable intake
of folate required to prevent spina bifida (Cuskelly et al. 1996).
Therefore, it is important to identify novel, naturally rich sources
of folate and to test that dietary strategies based on such foods
may be effective for the optimization of tissue folate in the general
population.
To assess the potential of ALF as a source of folate, it is
necessary to measure how much folate actually appears in the
blood after ingesting foods rich in this ingredient. To achieve,
this we performed a randomized, controlled intervention trial
to compare the change in plasma folate after consumption of
1) a cereal made from ALF, 2) a cereal made from wheat bran
(WB), and 3) a tablet containing 0. 5 mg folic acid that was
taken together with WB cereal.
MATERIALS AND METHODS
Sixteen healthy volunteers, eight males and eight females, aged
between 20 and 50 y, were recruited for this study, which was
approved by the Human Ethics Committee of Commonwealth Sci-
entific and Industrial Research Organisation Human Nutrition and
1
Supported by Goodman Fielder Milling and Baking Pty. Ltd.
2
To whom correspondence should be addressed.
3
Abbreviations used: ALF, aleurone flour; AUC, area the curve; RDI, recom-
mended dietary intake; WB, wheat bran.
0022-3166/99 $3.00 © 1999 American Society for Nutritional Sciences.
Manuscript received 7 December 1998. Initial review completed 21 January 1999. Revision accepted 26 February 1999.
1114
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complied with the Helsinki Declaration of 1975 as revised in 1983.
Volunteers who were supplementing their diet with folic acid and/or
who were deficient in plasma vitamin B-12 (,150 pmol/L) were
excluded from the study. The design of the study was a randomized,
controlled trial with a cross-over consisting of three intervention
rounds: 1) in the first round, each volunteer was randomly assigned to
the ALF cereal or the 0.5 mg folic acid tablet (Sigma Pharmaceuti-
cals, Victoria, Australia) with WB cereal; 2) in the second round,
all volunteers were given WB cereal only; and 3) in the third
round, there was a cross-over either to ALF cereal or 0.5 mg folic acid
tablet with WB cereal. The WB cereal was given as a low folate
control, and the 0.5 mg folic acid tablet with WB cereal was given as
a high folate positive control against which the ALF cereal could be
compared. The tablet was given together with WB cereal to provide
a dietary background identical to the low folate control. The WB
cereal was 100% extruded wheat bran, and the ALF cereal contained
90% aleurone flour and 10% waxy maize starch. It was estimated, by
microscopic image analysis, that 45% of the aleurone flour consisted
of aleurone cell contents (i.e., cytoplasm, nucleoplasm, and or-
ganelles) with the remainder (55%) consisting of aleurone cell walls.
The WB cereal and the ALF cereal were prepared by Goodman
Fielder Milling and Baking Pty. Ltd.
There were 7 d between each intervention round to allow
sufficient time for plasma folate to return to baseline before the
next round. On the day prior to each intervention round, volun-
teers were required to refrain from drinking alcohol and to fast
overnight. On the following morning volunteers donated a fasted
blood sample after which they ate 100 g of cereal with 250 ml fresh
milk (containing 1.5 g/100 g fat) over a period of 30 min. The folic
acid tablet was taken while the WB cereal was being ingested.
Further blood samples were collected at 1, 2, 4, and 7 h after
commencing cereal intake. During the course of the day, volun-
teers were provided with light snacks that were poor in folate (as
estimated from food composition tables, Holland et al. 1995), and
they were not allowed to eat any other foods. The level of folate
in the milk was 0.6
m
g/L. The texture and color of the ALF and
WB cereals was clearly different, but the volunteers were not
informed which of the cereals was made from ALF.
Plasma from coded blood samples collected in EDTA was isolated
immediately, snap frozen in liquid nitrogen, and stored at 280°C.
Plasma folate was measured using radioimmunoassay (BioRad Quan-
taphase II kit, Bio-Rad Laboratories Pty. Ltd. North Ryde, NSW,
Australia). Plasma vitamin B-12 measurements using radioimmuno-
assay (BioRad Quantaphase II kit; Bio-Rad Laboratories Pty. Ltd.
North Ryde, NSW, Australia) were also made as an internal analyt-
ical control. Samples were analyzed within 2– 4 wk after collection.
Under our conditions of frozen storage, there is no decline in plasma
folate for up to 8 mo of storage (data not shown). The assays for folate
and vitamin B-12 were performed in a single analysis on the same day,
and the intra-assay coefficients of variation were 5.5 and 4.8%,
respectively.
Coded samples of the ALF cereal, WB cereal, and tablets were
sent to the Australian Government Analytical Laboratories (South
Melbourne, Victoria) to measure the level of folate. The method used
was a modification of the standard Association of Official Analytical
Chemists microbiological assay with or without conjugase treatment
(Cunniff 1996, Scheelings 1996). Briefly the samples were milled in
a Cyclotec Sample Mill to a mesh size of ,0.5 mm. The samples were
then autoclaved in a phosphate buffer at 100°C for 5 min and then
cooled and filtered prior to deconjugation for3hatpH4.5with
human plasma (enzyme source) at 37°C. The enzyme reaction was
halted by boiling, the pH adjusted to 6.0, and an aliquot of the filtrate
was bioassayed with Lactobacillus casei var. rhamnosis as the culture.
Proximate analyses of wheat bran and aleurone flour were per-
formed. Total starch was analyzed using the method of McCleary et
al. (1994). Total dietary fiber was determined by the gravimetric
method of Prosky et al. (1985). Fat extraction was done according to
the method of Daugherty and Lento (1983), using Clarase digestion
followed by homogenization and extraction with chloroform/metha-
nol/water. The total nitrogen level was determined using the method
of Kirsten and Hesselius (1983) and a Carlo Erba Nitrogen Analyser
(Carlo Erba Strumentazione, Rodano, Milan). Free sugars were ex-
tracted with 80% aqueous methanol according to the method of
Theander and Westerlund (1986), and then quantified by HPLC
using acetonitrile:water 75:25 as the mobile phase, a Polyamine-
Bonded polymeric gel column, and a refractive index detector. Ash
was measured by igniting the samples in preweighed borosilicate glass
crucibles in a muffle furnace at 515°C for 12 h and then cooled in a
desiccator prior to weighing; the percentage ash weight was calcu-
lated by difference. The dry matter of the samples was determined by
drying the samples to constant weight at 110°C; they were then
cooled in a desiccator prior to weighing and moisture loss was calcu-
lated by difference.
The statistical significance of the changes in plasma folate follow-
ing ingestion of the WB and ALF cereals and the folic acid tablet was
determined using nonparametric, repeated measures ANOVA (Fried-
man test). The area under the plasma folate concentration versus
time curve [area under the curve (AUC)] was measured for each
individual and for each group at each intervention round using
plasma folate measured at0hasthebaseline value. The significance
of differences in the AUC values was also estimated using nonpara-
metric, repeated measures ANOVA and Dunn’s multiple comparison
test. All statistical analyses including measurements of AUC were
performed using PRISM software (GraphPad, San Diego, CA). All
quoted P-values are for two-tailed tests, unless otherwise indicated.
Differences were considered significant if P , 0. 05.
RESULTS
Analyses. Proximate analyses of the aleurone and wheat
bran flour indicated a higher starch and protein content and a
lower fiber content in aleurone flour when compared to wheat
bran flour (Table 1). The total folate level per 100 g in the
WB cereal and the ALF cereals was 94 6 4
m
g(n 5 2) and 515
6 7
m
g(n 5 2), respectively; the folic acid in each tablet was
526 6 24
m
g(n 5 3). The proportion of folate in the tablet,
ALF cereal, and WB cereal that could be detected without
prior treatment with folate conjugase was 100, 81, and 32%,
respectively.
Intervention. None of the volunteers were folate deficient
(plasma folate , 3.4 nmol/L). All volunteers completed the
intervention successfully. The results for plasma folate at each
time-point for each intervention round for males and females
are shown in Table 2, and the mean values (6 SEM) for the
combined results for males and females are shown graphically
in Fig. 2. There was a significant, positive correlation between
individual base-line data from each intervention round (R
5 0.69– 0.78, P , 0.001).
FIGURE 1 A schematic diagram showing the key steps in the
isolation of wheat bran and aleurone flour.
FOLATE FROM ALEURONE FLOUR 1115
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The plasma folate data during the WB cereal intervention
round clearly showed only a minimal increment in the vitamin
level during the course of the intervention; the increment was
significant in males only. In contrast, plasma folate levels
following ingestion of the 0.5 mg folic acid tablet with WB
cereal increased significantly in both males and females (P
5 0.003 and P 5 0.002, respectively); the combined results
showed a sharp increase in plasma folate during the first 2 h,
from a mean base-line level of 13.4 nmol/L to a peak at2hof
23.0 nmol/L, and a subsequent steady decline down to baseline
during the next 5 h (Fig. 2). The increase in plasma folate
following consumption of ALF cereal was also significant in
both males and females (P 5 0.0001) and appeared to be of
the same magnitude as that observed after the ingestion of 0.5
mg folic acid supplement with WB cereal, with a steady
increase in plasma folate during the first 2 h from a base-line
of 13.9 nmol/L to a peak at2hof23.5 nmol/L and a decline
down to baseline by 7 h (Fig. 2). However, the time response
following ALF cereal showed significant increments in plasma
folate at 2 and 4 h after ingestion of the cereal. This suggested
a slower rate of appearance of folate into the plasma compared
to the results for the folic acid tablet with WB cereal, which
showed significant increments in plasma folate at 1 and 2 h
following ingestion (Table 2). Analysis of the combined male
and female data showed that the observed increments in
plasma folate following intake of ALF cereal or folic acid tablet
with WB cereal were significant (P , 0.0001), but there was
no change following ingestion of the WB cereal.
To assess the extent of net folate appearance in the blood
we also measured the area under the plasma folate concentra-
tion versus time curve for each individual for each interven-
tion round. The folate AUC measured in the blood of all
subjects did not differ following ingestion of 100 g ALF cereal
[41.8 6 6.2 (nmol/L)h] or ingestion of the 0.5 mg folic acid
tablet with 100 g WB cereal [42.9 6 7.0 (nmol/L)h]; these
results were more than four times greater than the AUC
measured following the WB cereal intake [6.8 6 2.4 (nmol/
L)h] (Fig. 3). The same conclusions were reached when the
data for male (n 5 8, P , 0.01) and female (n 5 8, P , 0.05)
volunteers are analyzed separately; no significant effect of
gender on the AUC was detected for any treatment (Table 2).
To obtain a relative estimate of folate bioavailability, we
calculated the AUC/ingested folate ratios for the WB cereal,
TABLE 1
Proximate analysis of wheat bran flour and aleurone flour
1
Constituent Wheat bran flour Aleurone flour
g/100 g
Starch 21.6 36.5
Dietary fiber 31.6 15.4
Fat 5.2 6.5
Protein 17.8 23.6
Free sugars 6.2 7.2
Ash 3.5 4.1
Moisture 10.4 5.1
Sum 96.3 98.4
1
Values are means of duplicate analyses.
TABLE 2
Plasma folate at selected times after ingestion of wheat bran (WB) cereal, aleurone flour (ALF) cereal or 0.5 mg
folic acid tablet with WB cereal in men and women
1,2,3
Time, h
WB cereal ALF cereal 0.5 mg folic acid 1 WB cereal
Males Females Males Females Males Females
nmol/L
0 14.9 6 1.1 15.4 6 2.6 14.4 6 2.1 13.5 6 2.5 14.0 6 1.3 12.7 6 3.7
1 16.1 6 1.5 15.7 6 3.1 19.0 6 2.8 18.6 6 3.1 22.9 6 2.7* 20.8 6 2.9**
2 15.1 6 1.5 16.7 6 3.2 23.4 6 3.4** 23.6 6 4.1** 25.5 6 4.0** 20.5 6 2.9**
4 14.8 6 1.6 16.1 6 3.3 21.2 6 2.9** 21.6 6 3.7** 21.1 6 3.6 17.3 6 2.7
7 14.2 6 1.4 15.2 6 2.6 16.5 6 2.1 17.0 6 3.0 17.6 6 2.3 15.5 6 2.2
ANOVA P 0.029 0.680 0.0001 0.0001 0.003 0.002
AUC (nmol/L)h 5.7 6 3.2 8.0 6 3.8 38.1 6 6.8
†
45.6 6 10.6
‡
49.3 6 12.5
†
36.5 6 6.6
‡
1
Values are means 6 SEM; n 5 8.
2
ANOVA P-values refer to time effect; *P , 0.05, **P , 0.01 for comparison to means at time 0 h.
3
†P , 0.05,
‡
P , 0.01; significantly different from AUC after WB cereal consumption.
FIGURE 2 Change in plasma folate of men and women following
ingestion of wheat bran (WB) cereal, aleurone flour (ALF) cereal, and
0.5 mg folic acid with WB cereal. Results represent the mean 6 SEM, n
5 16, males and females combined. The ANOVA P-values for the
change in plasma folate with time for the WB cereal, ALF cereal, and 0.5
mg folic acid with WB cereal were 0.1139, ,0.0001, and ,0.0001, re-
spectively.
FENECH ET AL.1116
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the ALF cereal, and the folic acid tablet for each subject. The
bioavailability of folic acid from the tablet was calculated after
subtracting the AUC value for WB cereal from the AUC
value for the folic acid with WB cereal and then dividing by
the folic acid intake from the tablet only. The AUC/ingested
folate ratios for WB cereal, ALF cereal, and the folic acid
tablet of 0.0736 0.025, 0.081 6 0.012, and 0.068 6 0.012,
respectively, did not differ.
The individual AUC following ingestion of the ALF cereal
were not correlated with those observed after intake of the
tablet supplement with WB cereal, but they were positively
correlated with the AUC observed following intake of the WB
cereal (R 5 0.41, P 5 0.055, one-tailed test).
Significant positive correlations were observed between an
individual’s mean baseline value of plasma folate measured
before each intervention round and the AUC following in-
gestion of the WB cereal or the ALF cereal (R 5 0.53, P
5 0.03) (Fig. 4). However, this particular relationship was not
significant for AUC data from the folic acid tablet interven-
tion even though the correlation factor was positive (R
5 0.33, P 5 0.212). When the data for the ALF cereal
intervention were combined with the data for the tablet in-
tervention, the correlation coefficient was 0.427 (P 5 0.014).
Plasma vitamin B-12 concentrations remained constant
during the intervention (data not shown).
DISCUSSION
The lack of comprehensive and reliable information on the
concentration and bioavailability of folate from natural foods
has tended to favor the concept that fortification of staple
foods with synthetic folic acid is a more reliable strategy for
increasing tissue folate in the general population. It has re-
cently been shown, in a 3-mo intervention trial involving 41
women, that significant increments in red cell folate from an
initial mean value of 351
m
g/L to a postintervention value of
492
m
g/L were observed in those taking a supplement of 400
m
g/d synthetic folic acid, but there were no significant changes
in red cell folate in those who increased their intake of foods
rich in folate from a level that provided an estimated 209
m
g/d
to a level that was estimated to provide 410
m
g/d natural folate
(Cuskelly et al. 1996). In contrast, a randomized, placebo-
controlled trial designed to identify the minimum effective
dose for food fortification to prevent neural tube defects found
that a supplement of 200
m
g/d synthetic folic acid was suffi-
cient to raise red blood cell folate level above the 400
m
g/L
threshold (measured by microbiological bioassay) required for
minimization of spina bifida risk (Daly et al. 1997). In a similar
study, it was shown that 200
m
g/d of synthetic folic acid
appeared to be as effective as 400
m
g/d with regard to lowering
plasma homocysteine in apparently normal subjects (Ward et
al. 1997). An increase of 100
m
g/d of synthetic folic acid
intake was shown by both these studies to be relatively inef-
fective in terms of adequately reducing folate-related risk fac-
tors for spina bifida and cardiovascular disease.
The results from our study on cereal made from aleurone
flour show quite clearly that this natural source of folate can
make a significant difference in blood folate concentration. Of
main interest was 1) the much greater capacity for ALF cereal,
relative to WB cereal, to increase plasma levels of folate and 2)
that the increase in plasma folate following ingestion of 100 g
of ALF cereal was the same as that observed following intake
of 500
m
g synthetic folic acid with 100 g WB cereal. Our data
indicate that it is the higher content of aleurone flour rather
than increased bioavailability of folate from this product that
gives it value as a folate source in the diet.
Bailey et al. (1988) compared the bioavailability of mono-
glutamyl folate and polyglutamyl folate when ingested with
bran cereal and found that monoglutamyl folate bioavailability
is unaffected by dietary fiber, but wheat bran fiber appeared to
marginally inhibit the uptake of polyglutamyl folate. Using a
dual-label, stable isotope protocol, Pfeiffer et al. (1997)
showed that consuming folic acid with a light breakfast meal
only produced a small reduction in folic acid absorption (15%,
P . 0.05) relative to a control without food. Thus the com-
parison, in our study, between the AUC estimates following
ingestion of ALF cereal and folic acid in a tablet taken with
wheat bran cereal may have been influenced only to a limited
extent by fiber because 1) the tablet contained monoglutamyl
folate, and wheat bran only contributed a small percentage of
folate; and 2) the folate in the ALF cereal appeared to be in a
FIGURE 4 Relationship between base-line plasma folate and area
under curve (AUC) measurement in men and women after consumption
of the wheat bran (WB) cereal, aleurone flour (ALF) cereal, aleurone flour
(ALF) cereal, and 0. 5 mg folic acid with WB cereal. The correlation
factor for the relationship was 0.532 (P 5 0.034) for WB cereal, 0.529 (P
5 0.035) for ALF cereal, and 0.329 (P 5 0.212) for 0.5 mg folic acid with
WB cereal. The slope of the regression lines shown were only signifi-
cantly different from zero in the case of WB cereal (P 5 0.004) and ALF
cereal (P 5 0.024).
FIGURE 3 Plasma folate AUC following ingestion of wheat bran
(WB) cereal, aleurone flour (ALF) cereal, and 0.5 mg folic acid 1 WB
cereal. Results represent the mean 6 SEM, n 5 16, data for males and
females combined. Bars with different letters are significantly different
(P , 0.0001). AUC, area under curve, plasma folate concentration
versus time.
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relatively unconjugated form, and the level of fiber in the ALF
cereal was almost half that of the wheat bran cereal (Table 1).
The short-term, nonisotopic type of bioavailability study
used is very similar to that reported by Keagy et al. (1988) and
Bailey et al. (1988). In accordance with the recommendations
from these studies, multiple samples were taken over a 7-h
period to avoid errors in bioavailability estimation occurring
because of different rates of absorption. Bailey et al. (1988),
who measured plasma folate by microbiological assay, also
reported that with such a protocol it was only possible to
obtain consistent responses with doses of folic acid above 250
m
g—the folic acid/folate levels in the tablet with WB cereal
and the ALF cereal were well above this level. The WB cereal,
which contained only 92
m
g folate, produced a significant
increment in plasma folate only in males, suggesting that such
a level of ingested folate produces a change in plasma folate
that is at the limit of detection of the system used. Conse-
quently, the AUC measurement with the WB cereal may,
therefore, be considered less accurate than those obtained for
the ALF cereal and the folic acid tablet with WB cereal.
The apparent relative bioavailability of monoglutamyl and
polyglutamyl folates varies according to the protocol used
(Gregory 1995). For example, protocols with nonlabeled fo-
lates quantified by urinary excretion suggest a mean bioavail-
ability of 85–90% for tri- and hepta-glutamyl folate relative to
folic acid (Tamura and Stokstad 1973). Protocols with non-
labeled folate quantified by AUC of plasma folate concentra-
tion found equivalent bioavailability for 750
m
g folic acid and
the molar equivalent of heptaglutamyl folate, but the bioavail-
ability of the latter was reduced when given with bran cereal
but not spinach (Bailey et al. 1988). It is possible that in
certain cases the reported lower bioavailability of polyglutamyl
folate could have been caused by specific conjugase inhibitors
and not the extent of folate polyglutamation (Rosenberg and
Godwin 1971). Our results, obtained with similar techniques,
for wheat bran and aleurone flour, suggest no difference be-
tween the bioavailability of folate in these products and that
for folic acid in a tablet. Bioavailability of folate in the WB
and ALF flours may have been overestimated if the folate level
measured in the flours was underestimated by the single-
enzyme (conjugase) method used. However, it was shown that
for ready-to-eat cereals including wheat bran cereal, the level
of folate measured using the single-enzyme (conjugase)
method is the same as that measured by the tri-enzyme method
involving
a
-amylase, folate conjugase, and protease (Rader et
al. 1998). For other foods the tri-enzyme method yields a total
folate value that may be up to 30% higher than that deter-
mined by using conjugase alone (Rader et al. 1988).
The microbiological analyses in our study have shown that,
unlike folate in wheat bran, most of the folate in aleurone flour
could be detected without pretreatment with folate deconju-
gase enzyme. The reason for this difference is not known, but
it is possible that in the process of shearing aleurone cells
endogenous folate deconjugases are released and activated.
The shearing of aleurone cells may increase the bioavailability
of folate from this natural ingredient, particularly in those
individuals who have difficulty in digesting the thick cell walls
of aleurone cells. The apparent deconjugation of polygluta-
mate folate may make folate more available to people who
have limited deconjugase activity in the small intestine, pos-
sibly because of suboptimal pH levels, which may occur in
conditions such as atrophic gastritis, resulting in reduced gas-
tric acid secretion (Gregory 1995). Our estimates based on the
ratio of folate increments in the blood and ingested level of
folate suggest, however, that bioavailability of folate from ALF
cereal was not significantly greater than the bioavailability of
folate from WB cereal or synthetic folic acid from a tablet.
The results from this study give some indication that indi-
viduals with low baseline plasma folate seem to have lower
AUC for plasma folate than individuals with higher initial
plasma levels. The correlation factors suggest that between 9
and 25% of the observed variation in the AUC measurements
could be explained by baseline levels of plasma folate. The low
plasma folate and AUC levels may indicate either reduced gut
absorption or increased tissue uptake of folate from plasma.
Suboptimal jejunal pH may explain inefficient absorption of
folate in some cases (Gregory 1995, Russel et al. 1986). It may,
therefore, be useful in future studies to verify that blood folate
levels can be optimized by different strategies in those subjects
with an impaired capacity to absorb folate.
The time-related increment in plasma folate during the
initial 2 h occurred more rapidly following the ingestion of the
folic acid tablet with WB cereal than it did following ingestion
of ALF cereal. A conceivable explanation for the slower
uptake of folate from ALF cereal relative to folic acid from the
tablet is the necessity of folate deconjugation for the aleurone
source of folate. An alternative explanation could be that
folate from the ALF cereal was mainly absorbed by the jejunal,
pH-dependent, and saturable transport process, while absorp-
tion of folic acid from the tablet may have partly involved a
nonsaturable mechanism, such as passive diffusion (Gregory
1995, Halsted 1990, Mason 1990, Shoda et al. 1993, Strum
1979). It was reported that the latter mechanism may operate
when a bolus of synthetic folic acid of between 400 and 800
m
g
is ingested by humans and could result in substantial amounts
of unmetabolized folic acid appearing in plasma and urine
(Gregory 1995, Kelly et al. 1997, Lucock et al. 1989). In
contrast, physiological uptake by the saturable mechanism
into jejunal mucosal cells ensures that monoglutamyl folate is
reduced and methylated to form 5-methyltetrahydrofolate, the
major cytosolic folate in mammalian tissues, before transport
into the blood (Shane 1995). Therefore, it may be worthwhile
in future studies to compare the ratio of unmetabolized folic
acid and 5-methyltetrahydrofolate in plasma following inges-
tion of ALF cereal and a bolus of synthetic folic acid possibly
using established HPLC methods (Kelly et al. 1997, Lucock et
al. 1995).
Although the results from this study indicate that ALF
cereal is an important source of folate, long-term studies are
required to establish the extent to which folate from aleurone
flour may reduce plasma homocysteine and increase the level
of red cell folate, which is considered to be a reliable biomar-
ker of tissue folate stores. Such studies are currently underway
in our laboratory. Longer-term studies are also important be-
cause aleurone flour increases the rate of fermentation of
bacteria in the large bowel (Cheng et al. 1987). If ALF cereal
is favorable to the increase of folate-producing bacteria such as
Bifidobacteria (Krause et al. 1996), then there may be an
additional folate contribution via this route because recent
studies with rats and organ-cultured biopsies of human colon
suggest that folate can be absorbed across the large bowel
epithelium (Rong et al. 1991, Zimmerman 1990). These series
of potential events may explain the apparent positive associ-
ation between fiber intake and blood folate (Houghton et al.
1997). The significant contribution of folate from aleurone
cells may also explain in part why an increased intake of
whole-grain foods confers lowered risk for various digestive
tract cancers (Jacobs et al. 1998).
In conclusion, this study has shown that cereal made from
wheat aleurone flour is a significant source of natural, bioavail-
able folate that can make an effective contribution to increas-
FENECH ET AL.1118
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ing blood folate concentration. This effect is of a similar
magnitude to that observed following ingestion of 500
m
g
synthetic folic acid given with wheat bran cereal. These results
suggest that inclusion of foods made from wheat aleurone flour
in the diet can be considered as an alternative, important
strategy for increasing folate intake in the general population.
ACKNOWLEDGMENTS
We are grateful to Goodman Fielder Milling and Baking Pty. Ltd.
(Sydney, Australia) for providing the cereal products used in the
study. We would like to thank Josephine Rinaldi for very competently
performing the folate and vitamin B-12 measurements; Rodney
Trimble for sample processing, total dietary fiber, and free sugar
measurements; Sylvia Usher for the total starch measurements; Caro-
line Bignell for the total fat measurements; Ben Scherer; and Peter
Royle for the nitrogen measurements. We also thank Rosemary
McArthur and Clare Aitken for their important role in blood col-
lections and blood sample preparation for analysis, respectively.
LITERATURE CITED
Bailey, L. B. (1995) Folate requirements and dietary recommendations. In:
Folate in Health and Disease (Bailey, L. B., ed.), pp. 123–151. Marcel Dekker
Inc., New York, NY.
Bailey, L. B., Barton, L. E., Hillier, S. E. & Cerda, J. E. (1988) Bioavailability of
mono and polyglutamyl folate in human subjects. Nutr Rep Int. 38: 509–518.
Blount, B. C., Mack, M. M., Wehr, C. M., MacGregor, J. T., Hiatt, R. A., Wang, G.,
Wickramasinghe, S. N., Everson, R. B. & Ames, B. N. (1997) Folate
deficiency causes uracil misincorporation into human DNA and chromosome
breakage: Implications for cancer and neuronal damage. Proc. Natl. Acad.
Sci. 94: 3290–3295.
Boushey, C. J., Beresford, S. A., Omenn, G. S. & Motulsky, A. G. (1995) A
quantitative assessment of plasma homocysteine as a risk factor for vascular
disease: Probable benefits of increasing folic acid intakes. J. Am. Med. Assoc.
274: 1049–1057.
Cheng, B. Q., Trimble, R. P., Illman, R. J., Stone, B. A. & Topping, D. L. (1987)
Comparative effects of dietary wheat bran and its morphological components
(aleurone and pericarp-seed coat) on volatile fatty acid concentrations in the
rat. Br. J. Nutr. 57: 69–76.
Clysedale, F. M. (1994) Optimising the diet with whole grains. Crit. Rev. Food.
Sci. Nutr. 34: 453–471.
Crane, C. T., Wilson, D. B., Cook, D. A., Lewis, C. J., Yetley, E. A. & Rader, J. I.
(1995) Evaluating food fortification options: General principles revisited with
folic acid. Am. J. Pub. Health 85: 660–666.
Cunniff, P., ed. (1996) Official Methods Of Analysis Of AOAC International. Vol.
II. AOAC International, Gaithersburg, MA.
Cuskelly, G. J., McNulty, H. & Scott, J. M. (1996) Effect of increasing dietary
folate on red cell folate: Implications for prevention of neural tube defects.
Lancet 347: 657–659.
Czeizel, A. E. & Dudas, I. (1992) Prevention of first occurrence of neural tube
defects by periconceptional vitamin supplementation. N. Engl. J. Med. 327:
32–35.
Daly, S., Mills, J. L., Molloy, A. M., Conley, M., Lee, Y. J., Kirke, P. N., Weir, D. G.
& Scott, J. M. (1997) Minimum effective dose of folic acid for food fortifi-
cation to prevent neural tube defects. Lancet 350: 1666–1669.
Daugherty, C. E. & Lento, H. G. (1983) Chloroform-methanol extraction
method for determination of fat in foods: Collaborative study. J. Assoc. Off.
Anal. Chem. 66: 927–932.
Fenech, M. (1996) Folate & cancer initiation. Will folate fortification help to
prevent genetic events that could initiate cancer? Aust. J. Nutr. Diet. 53:
S13–S17.
Gregory, J. F. (1995) The bioavailability of folate. In: Folate in Health and
disease (Bailey, L. B., ed.), pp. 195–235. Marcel Dekker, New York, NY.
Halsted, C. H. (1990) Intestinal absorption of dietary folates. In: Folic Acid
Metabolism in Health and Disease (Picciano, M. F., Stokstad, E.L.R. & Greg-
ory, J. F., eds.), pp. 23–45. Wiley-Liss, New York, NY.
Helsinki Declaration (1996) Nuremberg Doctors’ Trial. Declaration of Helsinki.
B.M.J. 313: 1448–1449.
Holland, B., Welch, A. A., Unwin, I. D., Buss, D. H., Paul, A. A. & Southgate, D.A.T.
(1995) McCance and Widdowson’s: The composition of Foods, 5th ed.,
Xerox Ventura Publisher, Cambridge, UK.
Houghton, L. A., Green, T. J., Donovan, U. M., Gibson, R. S., Stephen, A. M., &
O’Connor D. L. (1997) Association between dietary fibre intake and the
folate status of a group of female adolescents. Am. J. Clin. Nutr. 66: 1414–
1421.
Jacobs, D. R., Marquart, L., Slavin, J. & Kushi, L. H. (1998) Whole-grain intake
and cancer: an expanded review and meta-analysis. Nutr. Cancer 30: 85–96.
Kang, S. S., Wong, P.W.K., Malinow, M. R. (1992) Hyperhomocyst(e)inemia as
a risk factor for occlusive vascular disease. Annu. Rev. Nutr. 12: 279–298.
Keagy, P. M., Shane, B. & Oace, S. M. (1988) Folate bioavailability in humans:
Effects of wheat bran and beans. Am. J. Clin. Nutr. 47: 80–88.
Kelly, P., McPartlin, J., Goggins, M., Weir, D. G. & Scott, J. M. (1997) Unme-
tabolised folic acid in serum: Acute studies in subjects consuming fortified
food and supplements. Am. J. Clin. Nutr. 65: 1790–1795.
Kirsten, W. J. & Hesselius, G. U. (1983) Rapid, automatic, high capacity Dumas
detection of nitrogen. Microchemical J. 28: 529–547.
Krause, L. J., Forsbery, C. W. & O’Connor, D. (1996) Feeding human milk to
rats increases Bifidobacterium in the cecum and colon which correlates with
enhanced folate status. J. Nutr. 126: 1505–1511.
Lucock, M. D., Green, M., Preistnall, M., Daskalakis, I., Levene, M. I. & Hartley, R.
(1995) Optimisation of chromatographic conditions for the determination of
folates in foods and biological tissues for nutritional and clinical work. Food
Chem. 53: 329–338.
Lucock, M., Wild, J., Smithells, R. & Hartley, R. (1989) Biotransformation of
pteroyl-monoglutamic acid during absorption: implications of Michaelis-Men-
ton kinetics. Eur. J. Clin. Nutr. 43: 631–635.
Ma, J., Stampfer, M. J., Giovannucci, E., Artigas, C., Hunter, D. J., Fuchs, C.,
Willett, W. C., Selhub, J., Hennekens, C. H. & Rozen, R. (1997) Methyl-
enetetrahydrofolate reductase polymorphism, dietary interactions and risk of
colorectal cancer. Cancer Res. 57: 1098–1102.
Mason, J. B. (1990) Intestinal transport of monoglutamyl folates in mammalian
systems. In: Folic Acid Metabolism in Health and Disease. (Picciano, M. F.,
Stokstad, E.L.R. & Gregory, J. F., eds.), pp. 47–64. Wiley -Liss, New York, NY.
McCleary, B. V., Solah, V. & Gibson, T. S. (1994) Quantitative measurement of
total starch in cereal flours and products. J. Cereal Sci. 20: 51–58.
Medical Research Council vitamin Study Research Group. (1991) Prevention
of neural tube defects: Results from the Medical Research Council Vitamin
Study. Lancet 338: 313–137.
Pfeiffer, C. M., Rogers, L. M., Bailey, L. M. & Gregory, J. F. III (1997) Absorption
of folate from fortified cereal-grain products and a supplemental folate con-
sumed with or without food determined by using a dual-label stable-isotope
protocol. Am. J. Clin. Nutr. 66: 1388–1397.
Prosky, L., Asp, N. G., Furda, I., DeVries, J. W., Schweizer, T. F. & Harland, B. F.
(1985) Determination of total dietary fibre in foods and food products:
Collaborative study. J. Assoc. Off. Anal. Chem. 68: 677–679.
Rader, J. I., Weaver, C. M. & Angyal, G. (1998) Use of a microbiological assay
with tri-enzyme extraction for measurement of pre-fortification levels of fo-
lates in enriched cereal-grain products. Food Chem. 62: 451–465.
Rong, N., Selhub, J., Goldin, B. R. & Rosenberg, I. H. (1991) Bacterially
synthesised folate in rat large intestine is incorporated into host tissue folyl
polyglutamates. J. Nutr. 121: 1955–1959.
Rosenberg, I. H. & Godwin, H. A. (1971) Inhibition of intestinal gamma-glutamyl
carboxypeptidase by yeast nucleic acid: An explanation of variability in utilisa-
tion of dietary polyglutamyl folate. J. Clin. Invest. 50:78a (abs.).
Russel, R. M., Kransinski, S. D., Samloff, I. M., Jacob, R. A., Hartz, S. C. &
Brovender, S. R. (1986) Folic acid malabsorption in atrophic gastritis.
Possible compensation by bacterial folate synthesis. Gastroenterology 91:
1476–1482.
Saxelby, C. & Venn-Brown, U. (1980) The structure and composition of the
wheat grain. In: The Role of Australian Flour and Bread in Health and Nutrition.
(Saxelby, C. and Venn-Brown, U. eds), pp. 37– 41. Glenburn Pty. Ltd., Chat-
swood, Australia
Scheelings, P. (1996) Prospects for measuring folates in Australian foods.
Aust. J. Nutr. Diet. 53: S23–S28.
Shane, B. (1995) Folate chemistry and metabolism. In: Folate in Health and
Disease. (Bailey, L. B., ed.), pp. 1–22. Marcel Dekker, New York, NY.
Shoda, R., Mason, J. B., Selhub, J. & Rosenberg, I. H. (1993) Folate binding in
intestinal brush border membranes. Evidence for two binding activities. J.
Nutr. Biochem. 1: 257–261.
Stenvert, N. (1995) New high fibre bread—Farrer’s Gold. Food Australia 47:
462–463.
Stenvert, N. (1997) Novel natural products from grain fractionation. In: Cere-
als—Novel Uses and Processes (Cambell, G. M., Webb, C & McKee, S. L.,
eds.), pp. 241–245. Plenum Press, New York, NY.
Strum, W. B. (1979) Enzymatic reduction and methylation of folate following
pH-dependent, carrier-mediated transport in jejunum. Biochim. Biophys. Acta
554: 249–257.
Subar, A. F., Block, G., & James, L. D. (1989) Folate intake and food sources
in the US population. Am. J. Clin. Nutr. 50: 508–516.
Tamura, T. & Stokstad, E.L.R. (1973) The availability of food folate in man.
Br. J. Haematol. 25: 513–512.
Theander, O. & Westerlund, E. (1986) Improved procedures for the analysis of
dietary fibre. J. Agric. Food Chem. 34: 330–336.
Ward, M., McNulty, H., McPartlin, J., Strain, J. J., Weir, D. G. & Scott, J. M.
(1997) Plasma homocysteine, a risk factor for cardiovascular disease, is
lowered by physiological doses of folic acid. QJM 90: 519–524.
Zimmerman, J. (1990) Folic acid transport in organ cultured mucosa of human
intestine. Gastroenterology 99: 964–972.
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