Comparative bioavailability of folate and vitamin C from a synthetic and a natural source1’ 2, 3 Edward
W. Nelson,4
M.D.,
ABSTRACT to study
was
jejunum.
Fifteen
predictably absorption
perfusion
water
the
of
over
studies,
measurement
of as their
synthetic
folate
absorption
previous
of absorption
not
amino
acids,
capacity.
and
Am.J.
C/in.
from first
the
the
30 cm can The
necessity
Nut,’.
28:
applied
urine
of triple
collections
in
3) it is a direct
1975.
cell ascorbate exceeded 20 g/ mm C saturation. were included intubated after
sent
Vitamin C was measured by the standard 2,4-dinitrophenyl hydrazine reaction modified by analysis with and without complete oxidation with copper ion (2). Results were expressed as total vitamin C and as oxidized products. The difference between the latter two analyses was expressed as L-ascorbic acid. Folic acid was measured by Lactobacillus casei bioassay-(3). The measurement of polyethelene glycol (PEG) was by turbidimetric analysis (4).
of these absorption
nutrients. are physio-
logical processes, occurring within the gastrointestinal tract, it seemed reasonable to approach this problem of human nutrition by direct measurement of intraluminal events. This report demonstrates that absorption of water-soluble vitamins can be quantitated from
a food
tube
perfusion,
source
can
nutrients tion
of their
Materials
triple
used a food
synthetic
to
relate
absorption
product
to
the
Denson
leukocytes
and
indicating
The four cigarette in this latter group. an overnight fast.
Bowers) adequate
smokers Each
(I) vita-
in the subject
study was
methods
lumen
previously applied of electrolytes, simple sugacids. Data obtained by this
be
from
intestinal
a method
to the absorption ars, and amino method
by
A nalvtic
108
of
and method,
The study of the comparative bioavailability of nutrients from food sources has relied on studies measuring urinary excretion and serum or cellular levels of these nutrients. It is not known whether serum and urinary levels of vitamin C and folic acid accurately repreintestinal absorption Since digestion and
(method
the
solutions.
This
and
1014-1019,
two
be reliably
for
a
solution,
advantages
of absorption,
acid
Despite
ofjejunum
absorption.
sources.
study
proximal of folic
juice
electrolytes,
mechanism
human vitamins.
orange
water
food
extensively
a 30-cm
absorption
in the
and
from
1) it overcomes sites
the
significantly with
used
with
of synthetic
absorbed
positively
been
tube
from
containing
differ
avidly
M.D.
not
to quantitate
a solution
of nutrients are:
vitamins
controls
glucose
were
to study
lumen
the
correlated
methods
has
a triple
from
ofsugars,
be used
intestine
study
and did
folate
bioavailability
2) it can
own
J. Cerda,6
small
water-soluble
vitamins and
to the
In this
from
ascorbate
and James
human
solution
absorption
of comparative
perfusion
metabolic
juice
water-soluble
synthetic applied
study
served
orange
two
of the
absorption
subjects
greater
M.D.
of vitamins.
measure
normal
exception
previously lumen
to an
and the
bioavailability
C from of
Natural
to the
Intraluminal used
vitamin
with
R. Streiff5
comparative
segment and
Richard
of absorp-
counterparts.
and methods
I From the Department of Medicine, University of Florida College of Medicine and the Veterans Administration Hospital, Gainesville, Florida 32601. 2 Address reprint requests to: Edward W. Nelson, M.D., Division of Gastroente,rology, Box 230, J. Hillis
Miller
Health Center, Gainesville, Florida Supported by a grant from the Commission, Lakeland, Florida. Presented Western Hemisphere Nutrition Congress
32610. Florida Citrus in part at the IV, August 20,
1974,
Subjects
The
study involved 15 normal subjects on an adequate diet, age 20-42 years with a mean of 24 years. In all of the ten subjects in whom it was measured, white blood
1014
The
American
Journal
of Clinical
Nutrition
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/9/1014/4716425 by University of Glasgow user on 03 April 2018
Bal Harbour, Florida. Assistant Professor, Division of terology. 5Chief, Medical Service, Veterans tration Hospital, Gainesville, Florida. Professor, Division of Gastroenterology.
28:
SEPTEM
BER
1975,
pp.
1014-
1019.
Printed
GastroenAdminisAssociate
in U.S.A.
COMPARATIVE Intestinal
BIOAVAILABILITY
part to 7 parts water and a synthetic solution containing ascorbic acid and folic acid (both Sigma Chemical Co., St. Louis, Mo.) in concentrations identical to the natural orange juice perfusate. The composition of the solutions is listed in Table I. Both solutions were approximately isotonic, pH of 7.0 and temperature of 37 C when infused. Both solutions were perfused by means of a Harvard infusion pump at 458 mI/hour. Aspiration of the proximal and distal ports was performed with Wappler-Stedman model no. 2590 (A.C.M.I., New York) pumps at 2 ml/min. Samples were collected in five consecutive 10-mm aliquots. The collections were “staggered” such that the distal aspirate was compared with the proximal aspirate of the previous 10-mm period thus allowing for 10-mm solution transit time in the mixing segment. A 30-mm equilibration period preceded the first collection and a 60-mm perfusion period separated the two solutions in a tandem study (6). As nearly as possible, a steady state of perfusion, aspiration, and absorption was maintained. Calculations
and
statistical
Results absorption
Figure 1 demonstrates the comparison of water absorption in the 15 subjects, each perfused with both solutions and serving as his own control. The orange juice solution
Composition
AND
VITAMIN
1015
C
150
I00 I
C
50
FIG. 1. orange
showed water
of solutions
perfused
in each
subject’
Water absorption (hatched bars)
Results expressed from synthetic than from orange
a
in 15 subjects and synthetic
as mean ± SEM. solution significantly juice solution.
statistically
significant
(96 (58
absorption
perfused (clear
Water (P
0.05, t 0.388). Owing to variations in orange juice vitamin C concentration and to =
absorption
of
the
vitamin
from
7.0
when
perfused.
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/9/1014/4716425 by University of Glasgow user on 03 April 2018
the
mixing segment, a larger amount of synthetic vitamin C ( 50 ± 5 mg) than orange juice vitamin C (z 39 ± 3.5) entered the study segment. Thus, the percent vitamin C absorbed from orange juice (s 40 ± 3.5%) was somewhat higher than the percent vitamm C absorbed from the synthetic (j 36 ± 2.8%) but that difference did not achieve statistical significance (P > 0.05, t 0.06). The percent absorption of vitamin C was =
=
=
=
pH
the
related positively with the absorption of natural vitamin C (r 0.653, a 0.05) and natural folate (r 0.636, a 0.05) as well as synthetic vitamin C (r 0.686, a 0.05) but not with synthetic folate (r -0.05). The data are represented in Fig. 2.
=
Orange
with bars)
synthetic solution ± 1 1 ml, P < 0.05). Nine of the fifteen subjects demonstrated greater water absorption with the natural
greater
I
juice
solutions. absorption 0.05) less
orange
anal vsis
Solute and water absorption were calculated by standard formulas and expressed as amount absorbed (ng or mg/cm per hr) or as percent absorbed (6). The latter value represents the amount of solute absorbed from the study segment divided by the amount entering the segment per unit time. The results were analyzed by both the Student’s t-test and by the paired difference test (7). Correlation of water and nutrient absorption was by the method of least squares (7).
TABLE
FOLATE
perfusion
The method of triple lumen intestinal perfusion was adapted from that of Inglefinger et al. (5) utilizing a 15-cm mixing segment and a 30-cm study segment. The tube was positioned fluoroscopically in each subject so that the infusion port was placed at the Ligament of Treitz. The two solutions perfused in the experiment consisted of frozen orange juice concentrate diluted I
Water
OF
NELSON
1016 greater
from the orange juice mixture in 8 of subjects. There was no statistical in the absorption of vitamin C the 4 cigarette smokers and I I non-
15 difference between smokers. the
Folic
acid
absorption
Figure 4 illustrates tion of folic acid (orange juice z L 12 ± 0.7 Lg,
nearly identical absorpfrom the two solutions 1 1 .8 ± 0.7 tg, synthetic P > 0.1, t 0.25). Since
=
=
the
folic
amounts
of
=
orange
juice
and
acid
entering the study ;sg and 19.4 /Lg, respectively) equal, the respective percent ± 4.5% and 58 ± 3%) was equal (P > 0. 1, t 0).
synthetic
segment were absorption likewise
(18.4 nearly (60 nearly
=
Discussion The problem bility of natural been approached
of the comparative bioavailaand synthetic vitamin C has by several investigators with conflicting results. The comparison of serum and urine levels of ascorbic acid after oral intake of comparable doses of natural and synthetic vitamins have led to the conclusion that natural vitamin C shows greater (8), equal (9), or less (10) availability than a synthetic source of the vitamin. The latter report concluding inferior bioavailability of the
natural
vitamin
stated
in “bioavailability” from
that
apparently
differences
in
the
difference
did
not
absorption
rate
but
result was
partly because of a greater urinary excretion oftotal ascorbic acid after ingestion of orange juice (10). Such a conclusion underscores the difficulty of what precisely is included in the term,
lism the
bioavailability,
and definition.
i.e.,
excretion
are
Bioavailability
whether
metabo-
to be included as measured
within by
intestinal perfusion techniques reflects uptake ofthe vitamin and the data so obtained do not indicate significant differences in intestinal uptake of vitamin C from orange juice or from a synthetic source. Streiff (I 1 ) has found that citrus products are a rich source of folate and has emphasized that citrus folate is not altered by cooking or processing as is folate in many other food sources. Because citrus products contain folic acid almost exclusively in the monoglutamate form, and there is evidence that this form is most readily absorbed (12), it would be
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/9/1014/4716425 by University of Glasgow user on 03 April 2018
ET AL.
anticipated that orange juice folate should be avidly absorbed. A recently reported metabolic balance study concluded, however, that orangejuice folate was characterized by a low level of bioavailability as determined by unnary excretion after a loading dose (13). Almost identical results of transport rate and percentage absorption of folic acid from orange juice and synthetic sources in this study are contrary to these results and mdicate that orange juice folate is equal in bioavailability to synthetic pteroylglutamic acid. Another aspect of this study was to determine whether the site of absorption in the gastrointestinal tract would differ for food sources and synthetic sources of water-soluble vitamins. Both sources show similar patterns of absorption in the proximal jejunum with perfusion of this area. Nicholson and Chornock (14) employed an intestinal perfusion method 32 years ago to demonstrate that vitamin C is absorbed pnincipally in the proximal jejunum. The perfusion recovery method used in that study has since been shown to overestimate intestinal absorption since 100% recovery of perfusate and intestinal secretion is not possible by this method (15). Thus, their observed mean absorption of synthetic vitamin C (48%) exceeds the mean of 36% in this study for a comparable infused load. The conclusion that a substantial portion of perfused vitamin C is absorbed in the first 30 cm of jejunum is supported by both methods. Recently, it has been demonstrated that L-ascorbic acid may be absorbed by an active transport process in the distal ileum of both the guinea pig and man (16). Low values of serum, leukocyte and tissue levels of ascorbic acid have been observed in patients with regional entenitis involving the terminal ileum and to be associated with a clinical complication of this disease, fistula formation (17). The demonstration that there is avid absorption of vitamin C in the proximal jejunum does not exclude the terminal ileum as an important reservoir of absorptive function. Presently, studies are in progress to determine whether absorption of vitamin C in the jejunum is characterized by saturation kinetics with increasing loads, thereby suggesting a carrier-mediated mechanism at this site. The proximal jejunum has been docu-
COMPARATIVE
OF FOLATE
BIOAVAILABILITY
AND
VITAMIN
1017
C
220 B r - 0 636 0653
200
ISO
S
I
.
60
60
S
40
40
120
20
S
I C
00
.
80 .
60 .
40
/
S. S S
S
.2
4
6
8
0
200
12
400
600
800
K100
ISO
80 C
D
r._005
06$6
60
S
160
S
40
40
I 20
120
S
S
100
I00
S
S
S
80
80
E
S
S
60
60
40
40
S
20
20 S
S
S
0
20 4
2
6
8
0
.4
2
200
400
mg/cm/HR
FIG. synthetic are
2. Correlation between water vitamin C, and D: synthetic
significantly
(a
0.05)
different
6bo
800
I000
ng/cm/HR
absorption folate with from
and least
transport squares
rate of A: natural regression lines. The
vitamin C, correlations
B: natural folate, C: for sets A, B, and C
zero.
mented by several investigators to be the principal site of absorption of synthetic folic acid (18, 19). A conclusion of this study is that food sources of folic acid probably do not
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/9/1014/4716425 by University of Glasgow user on 03 April 2018
differ as to site of absorption from sources. The figure of approximately absorption of folic acid in dietary from the proximal jejunum agrees
synthetic 60% amounts well with
NELSON
1018
20
IS
+
75
+
;
U)
a 4 C (0 -5
50 -4 10
-I-
-5 a
25 5
FIG. 3. Absorption of vitamin C as total amount and as percentage absorption in the 30-cm study segment from orange juice (hatched bars) and synthetic (clear bars) solutions. Means ± SEM indicated. Neither parameter shows statistically significant (P 0.05) difference. I
00
0
4
8C
Previous investigations have shown that glucose can enhance folate absorption (20). The absorption of water-soluble vitamins, ascorbic acid and folic acid did not differ in the two solutions despite a large difference in water absorption; this result, however, may be explained as follows. The orange juice solution may contain a “binder” which prevents augmentation of absorption of folate and vitamin C, despite enhanced water transport. Thus, if glucose were present in the synthetic mixture, the absorption of the waten-soluble vitamins might be increased over that from the orange juice mixture. The lack of correlation of synthetic folate absorption with water absorption contrasts with the positive correlation of water and vitamin C absorption. Differences in mode of transport, i.e., active transport versus passive diffusion, might be one explanation. Currently, studies are in progress to evaluate these possibilities. It is the conclusion of this study that there
is avid
IS
C
ET AL.
0
U
FIG. 4. Absorption of folic acid as total amount and as percentage absorption in the 30-cm study segment from orange juice (hatched bars) and synthetic (clear bars) solutions. Means ± SEM indicated. Neither parameter shows statistically significant (P 0.05) difference.
absorption
of vitamin
C and
folic acid
in the proximal jejunum from a synthetic and a food (citrus) source. These sources did not differ significantly in the availability of these nutrients. We conclude, also, that the method of triple lumen intestinal perfusion can quantitate absorption of water-soluble vitamins from food sources in human volunteers and that such data can be used to relate absorption of nutrients from a food product to the absorption of their synthetic counterparts. The method, then, is useful as applied to the problems of comparative bioavailability in human nutrition. The authors express appreciation Green, Mrs. Wanda Durrance, Mrs. and Mrs. Muriel Hogan for their
to Miss Bonnye Eugenia Ahmed, expert technical
assistance.
the data of Butterworth et al. (19), but not with that of Gerson et al. (20). The observed results of augmented water absorption with the orange juice mixture are consistent with previous data that glucose in concentrations of 14 mrs (240 mg/l00 ml) or greater promotes absorption of Nat, C1 and water (21). Maximum rates of water absorption are obtained from isotonic solutions containing approximately 56 mM (or 1%) glucose. The orangejuice solution used in this study contained 1.2% glucose; therefore, enhanced water absorption was expected.
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/9/1014/4716425 by University of Glasgow user on 03 April 2018
References 1. DENSON K. W. AND E. F. BOWERS. The determination of ascorbic acid in white blood cells. Clin. Sci. 21: 157-162, 1961. 2. BESSEl, 0. A., 0. H. LOWRY AND M. D. BROCK. The quantitative determination of ascorbic acid in small amounts of white blood cells and platelets. J. Biol. Chem. 168: 197, 1947. 3. HERBERT, V. Aseptic addition method for Laciobacillus casei assay of folate activity in human serum. J. Clin. Pathol. 19: 12-16, 1966. 4. MALAWER, S. D. AND D. W. POWELL. An improved turbidimetric analysis of polyethylene glucol utilizing an emulsifier. Gastroenterology 53: 250-256,
COMPARATIVE 1967. 5. CooPER, H., R. LEVITAN, INGELFINGER. A method water and solute from Gastroenterology
6.
K. H.
50:
SORGEL.
the human perfusion
1 -7, and
J. E.
water
GEENEN
absorption
intestine. The Gastroenterology
AND
accuracy 51:
of the 975-984,
1966. G. W.
7. SNEDECOR,
Methods, 8.
A.
ELMBY,
W. G.
AND
Ames:
Iowa
E.
AND
State
COCHRAN
Statistical
Univ.
Press, 1967. The inadequacy of antiscorbutic agent.
WARBURG.
synthetic ascorbic acid as an Lancet 11: 1363-1365, 1937. 9. TODHUNTER, E. N. AND A. S. FATZER. A comparison of the utilization by college women of equivalent amounts of ascorbic acid in red raspberries and crystalline form. J. Nutr. 19: 1 13-130, 1940.
10.
synthetic
Assoc.
11.
and
HALSTED,
in citrus 1390-1392,
ity of food folate 513-531, 1973.
19.
et.
BAUGH
AND
and other 1971.
C. E.
E. L. in
STAKSTACE.
man.
Brit.
The
J.
20.
21. availabil-
25:
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/9/1014/4716425 by University of Glasgow user on 03 April 2018
D. C.
method
of intraluminal Digestion 9:176-192,
RAMBAUD
fistula formation 67: 428-433,
D.
AND
D.
GERSON,
HERBERT
absorption in Gastroenterology
SLADEN
ships water 119,
C. D., N. V.
G. E., between by the 1969.
AND
in man.
M.
perfusion 1973.
in regional 1974.
C. W., C. C. BooTH, J. AND D. L. MOLLIN.
crystalline folic acid 1968. HALSTED, C. H., C. WORTH Jejunal uptake Gastroenterology 66:
acid cose.
in man.
Haematol.
HEPNER,
BROWN,
BUTTER-
uptake of conjugated folate 66: 706, 1974 (abstr.). AND
deficiency and Gastroenterology
in
Diet
R.,
small
HOFFBRAND
of
J. Am.
The
of intestine. 16. STEVENSON, N. R. AND M. K. BRUSH. The existence and characterization of Nat-dependent active transport ofascorbic acid in guinea pig. Am J. Clin. Nuts. 22: 318-326. 1969. 17. GERSON, C. D., AND E. M. FABRY. Ascorbic acid the
1974.
C. H., C. M.
T,
MODIGLIANI,
18.
Bioavailability
acid.
Folate levels J. Clin. Nutr. 24:
Jejunal Gastroenterology TAMUROD,
KEITH.
ascorbic
R. R. Am.
WORTH.
13.
M.
AND
natural
64: 271-275,
STREIFF,
juices. 12.
0.
PELLETIER,
1019
C
15.
BERNIER.
from
VITAMIN
NICHOLSON, D. T., AND F. W. CHORNOCK. Intubation studies of the human small intestine. An improved technic for the study of absorption; its application to ascorbic acid. J. Clin. Invest. 21: 505, 1942.
1966.
HARRIS,
AND
14. FORDTRAN AND F. I. of studying absorption of the human small intestine.
Sodium
small method.
OF FOLATE
D. S.
G. E., D. A.
WHALEN,
BIOAVAILABILITY
Lancet
BAUGH
AND
AND
man:
Absorption
A. M.
of
I I: 302-306,
C. E.
BUTTER-
in man.
G. W. HEPNER, H. D. JANOWITZ.
Enhancing 61: 224-227,
the absorption normal human
A. V.
COWAN,
of conjugated folate 706, 1974 (abstr.). COHEN,
enteritis.
DAWSON.
of glucose, jejunum.
effects 1971.
N. Folic
of glu-
Interrelation-
sodium, and Clin. Sci. 36:
W. Nelson,4
M.D.,
ABSTRACT to study
was
jejunum.
Fifteen
predictably absorption
perfusion
water
the
of
over
studies,
measurement
of as their
synthetic
folate
absorption
previous
of absorption
not
amino
acids,
capacity.
and
Am.J.
C/in.
from first
the
the
30 cm can The
necessity
Nut,’.
28:
applied
urine
of triple
collections
in
3) it is a direct
1975.
cell ascorbate exceeded 20 g/ mm C saturation. were included intubated after
sent
Vitamin C was measured by the standard 2,4-dinitrophenyl hydrazine reaction modified by analysis with and without complete oxidation with copper ion (2). Results were expressed as total vitamin C and as oxidized products. The difference between the latter two analyses was expressed as L-ascorbic acid. Folic acid was measured by Lactobacillus casei bioassay-(3). The measurement of polyethelene glycol (PEG) was by turbidimetric analysis (4).
of these absorption
nutrients. are physio-
logical processes, occurring within the gastrointestinal tract, it seemed reasonable to approach this problem of human nutrition by direct measurement of intraluminal events. This report demonstrates that absorption of water-soluble vitamins can be quantitated from
a food
tube
perfusion,
source
can
nutrients tion
of their
Materials
triple
used a food
synthetic
to
relate
absorption
product
to
the
Denson
leukocytes
and
indicating
The four cigarette in this latter group. an overnight fast.
Bowers) adequate
smokers Each
(I) vita-
in the subject
study was
methods
lumen
previously applied of electrolytes, simple sugacids. Data obtained by this
be
from
intestinal
a method
to the absorption ars, and amino method
by
A nalvtic
108
of
and method,
The study of the comparative bioavailability of nutrients from food sources has relied on studies measuring urinary excretion and serum or cellular levels of these nutrients. It is not known whether serum and urinary levels of vitamin C and folic acid accurately repreintestinal absorption Since digestion and
(method
the
solutions.
This
and
1014-1019,
two
be reliably
for
a
solution,
advantages
of absorption,
acid
Despite
ofjejunum
absorption.
sources.
study
proximal of folic
juice
electrolytes,
mechanism
human vitamins.
orange
water
food
extensively
a 30-cm
absorption
in the
and
from
1) it overcomes sites
the
significantly with
used
with
of synthetic
absorbed
positively
been
tube
from
containing
differ
avidly
M.D.
not
to quantitate
a solution
of nutrients are:
vitamins
controls
glucose
were
to study
lumen
the
correlated
methods
has
a triple
from
ofsugars,
be used
intestine
study
and did
folate
bioavailability
2) it can
own
J. Cerda,6
small
water-soluble
vitamins and
to the
In this
from
ascorbate
and James
human
solution
absorption
of comparative
perfusion
metabolic
juice
water-soluble
synthetic applied
study
served
orange
two
of the
absorption
subjects
greater
M.D.
of vitamins.
measure
normal
exception
previously lumen
to an
and the
bioavailability
C from of
Natural
to the
Intraluminal used
vitamin
with
R. Streiff5
comparative
segment and
Richard
of absorp-
counterparts.
and methods
I From the Department of Medicine, University of Florida College of Medicine and the Veterans Administration Hospital, Gainesville, Florida 32601. 2 Address reprint requests to: Edward W. Nelson, M.D., Division of Gastroente,rology, Box 230, J. Hillis
Miller
Health Center, Gainesville, Florida Supported by a grant from the Commission, Lakeland, Florida. Presented Western Hemisphere Nutrition Congress
32610. Florida Citrus in part at the IV, August 20,
1974,
Subjects
The
study involved 15 normal subjects on an adequate diet, age 20-42 years with a mean of 24 years. In all of the ten subjects in whom it was measured, white blood
1014
The
American
Journal
of Clinical
Nutrition
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/9/1014/4716425 by University of Glasgow user on 03 April 2018
Bal Harbour, Florida. Assistant Professor, Division of terology. 5Chief, Medical Service, Veterans tration Hospital, Gainesville, Florida. Professor, Division of Gastroenterology.
28:
SEPTEM
BER
1975,
pp.
1014-
1019.
Printed
GastroenAdminisAssociate
in U.S.A.
COMPARATIVE Intestinal
BIOAVAILABILITY
part to 7 parts water and a synthetic solution containing ascorbic acid and folic acid (both Sigma Chemical Co., St. Louis, Mo.) in concentrations identical to the natural orange juice perfusate. The composition of the solutions is listed in Table I. Both solutions were approximately isotonic, pH of 7.0 and temperature of 37 C when infused. Both solutions were perfused by means of a Harvard infusion pump at 458 mI/hour. Aspiration of the proximal and distal ports was performed with Wappler-Stedman model no. 2590 (A.C.M.I., New York) pumps at 2 ml/min. Samples were collected in five consecutive 10-mm aliquots. The collections were “staggered” such that the distal aspirate was compared with the proximal aspirate of the previous 10-mm period thus allowing for 10-mm solution transit time in the mixing segment. A 30-mm equilibration period preceded the first collection and a 60-mm perfusion period separated the two solutions in a tandem study (6). As nearly as possible, a steady state of perfusion, aspiration, and absorption was maintained. Calculations
and
statistical
Results absorption
Figure 1 demonstrates the comparison of water absorption in the 15 subjects, each perfused with both solutions and serving as his own control. The orange juice solution
Composition
AND
VITAMIN
1015
C
150
I00 I
C
50
FIG. 1. orange
showed water
of solutions
perfused
in each
subject’
Water absorption (hatched bars)
Results expressed from synthetic than from orange
a
in 15 subjects and synthetic
as mean ± SEM. solution significantly juice solution.
statistically
significant
(96 (58
absorption
perfused (clear
Water (P
0.05, t 0.388). Owing to variations in orange juice vitamin C concentration and to =
absorption
of
the
vitamin
from
7.0
when
perfused.
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/9/1014/4716425 by University of Glasgow user on 03 April 2018
the
mixing segment, a larger amount of synthetic vitamin C ( 50 ± 5 mg) than orange juice vitamin C (z 39 ± 3.5) entered the study segment. Thus, the percent vitamin C absorbed from orange juice (s 40 ± 3.5%) was somewhat higher than the percent vitamm C absorbed from the synthetic (j 36 ± 2.8%) but that difference did not achieve statistical significance (P > 0.05, t 0.06). The percent absorption of vitamin C was =
=
=
=
pH
the
related positively with the absorption of natural vitamin C (r 0.653, a 0.05) and natural folate (r 0.636, a 0.05) as well as synthetic vitamin C (r 0.686, a 0.05) but not with synthetic folate (r -0.05). The data are represented in Fig. 2.
=
Orange
with bars)
synthetic solution ± 1 1 ml, P < 0.05). Nine of the fifteen subjects demonstrated greater water absorption with the natural
greater
I
juice
solutions. absorption 0.05) less
orange
anal vsis
Solute and water absorption were calculated by standard formulas and expressed as amount absorbed (ng or mg/cm per hr) or as percent absorbed (6). The latter value represents the amount of solute absorbed from the study segment divided by the amount entering the segment per unit time. The results were analyzed by both the Student’s t-test and by the paired difference test (7). Correlation of water and nutrient absorption was by the method of least squares (7).
TABLE
FOLATE
perfusion
The method of triple lumen intestinal perfusion was adapted from that of Inglefinger et al. (5) utilizing a 15-cm mixing segment and a 30-cm study segment. The tube was positioned fluoroscopically in each subject so that the infusion port was placed at the Ligament of Treitz. The two solutions perfused in the experiment consisted of frozen orange juice concentrate diluted I
Water
OF
NELSON
1016 greater
from the orange juice mixture in 8 of subjects. There was no statistical in the absorption of vitamin C the 4 cigarette smokers and I I non-
15 difference between smokers. the
Folic
acid
absorption
Figure 4 illustrates tion of folic acid (orange juice z L 12 ± 0.7 Lg,
nearly identical absorpfrom the two solutions 1 1 .8 ± 0.7 tg, synthetic P > 0.1, t 0.25). Since
=
=
the
folic
amounts
of
=
orange
juice
and
acid
entering the study ;sg and 19.4 /Lg, respectively) equal, the respective percent ± 4.5% and 58 ± 3%) was equal (P > 0. 1, t 0).
synthetic
segment were absorption likewise
(18.4 nearly (60 nearly
=
Discussion The problem bility of natural been approached
of the comparative bioavailaand synthetic vitamin C has by several investigators with conflicting results. The comparison of serum and urine levels of ascorbic acid after oral intake of comparable doses of natural and synthetic vitamins have led to the conclusion that natural vitamin C shows greater (8), equal (9), or less (10) availability than a synthetic source of the vitamin. The latter report concluding inferior bioavailability of the
natural
vitamin
stated
in “bioavailability” from
that
apparently
differences
in
the
difference
did
not
absorption
rate
but
result was
partly because of a greater urinary excretion oftotal ascorbic acid after ingestion of orange juice (10). Such a conclusion underscores the difficulty of what precisely is included in the term,
lism the
bioavailability,
and definition.
i.e.,
excretion
are
Bioavailability
whether
metabo-
to be included as measured
within by
intestinal perfusion techniques reflects uptake ofthe vitamin and the data so obtained do not indicate significant differences in intestinal uptake of vitamin C from orange juice or from a synthetic source. Streiff (I 1 ) has found that citrus products are a rich source of folate and has emphasized that citrus folate is not altered by cooking or processing as is folate in many other food sources. Because citrus products contain folic acid almost exclusively in the monoglutamate form, and there is evidence that this form is most readily absorbed (12), it would be
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ET AL.
anticipated that orange juice folate should be avidly absorbed. A recently reported metabolic balance study concluded, however, that orangejuice folate was characterized by a low level of bioavailability as determined by unnary excretion after a loading dose (13). Almost identical results of transport rate and percentage absorption of folic acid from orange juice and synthetic sources in this study are contrary to these results and mdicate that orange juice folate is equal in bioavailability to synthetic pteroylglutamic acid. Another aspect of this study was to determine whether the site of absorption in the gastrointestinal tract would differ for food sources and synthetic sources of water-soluble vitamins. Both sources show similar patterns of absorption in the proximal jejunum with perfusion of this area. Nicholson and Chornock (14) employed an intestinal perfusion method 32 years ago to demonstrate that vitamin C is absorbed pnincipally in the proximal jejunum. The perfusion recovery method used in that study has since been shown to overestimate intestinal absorption since 100% recovery of perfusate and intestinal secretion is not possible by this method (15). Thus, their observed mean absorption of synthetic vitamin C (48%) exceeds the mean of 36% in this study for a comparable infused load. The conclusion that a substantial portion of perfused vitamin C is absorbed in the first 30 cm of jejunum is supported by both methods. Recently, it has been demonstrated that L-ascorbic acid may be absorbed by an active transport process in the distal ileum of both the guinea pig and man (16). Low values of serum, leukocyte and tissue levels of ascorbic acid have been observed in patients with regional entenitis involving the terminal ileum and to be associated with a clinical complication of this disease, fistula formation (17). The demonstration that there is avid absorption of vitamin C in the proximal jejunum does not exclude the terminal ileum as an important reservoir of absorptive function. Presently, studies are in progress to determine whether absorption of vitamin C in the jejunum is characterized by saturation kinetics with increasing loads, thereby suggesting a carrier-mediated mechanism at this site. The proximal jejunum has been docu-
COMPARATIVE
OF FOLATE
BIOAVAILABILITY
AND
VITAMIN
1017
C
220 B r - 0 636 0653
200
ISO
S
I
.
60
60
S
40
40
120
20
S
I C
00
.
80 .
60 .
40
/
S. S S
S
.2
4
6
8
0
200
12
400
600
800
K100
ISO
80 C
D
r._005
06$6
60
S
160
S
40
40
I 20
120
S
S
100
I00
S
S
S
80
80
E
S
S
60
60
40
40
S
20
20 S
S
S
0
20 4
2
6
8
0
.4
2
200
400
mg/cm/HR
FIG. synthetic are
2. Correlation between water vitamin C, and D: synthetic
significantly
(a
0.05)
different
6bo
800
I000
ng/cm/HR
absorption folate with from
and least
transport squares
rate of A: natural regression lines. The
vitamin C, correlations
B: natural folate, C: for sets A, B, and C
zero.
mented by several investigators to be the principal site of absorption of synthetic folic acid (18, 19). A conclusion of this study is that food sources of folic acid probably do not
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differ as to site of absorption from sources. The figure of approximately absorption of folic acid in dietary from the proximal jejunum agrees
synthetic 60% amounts well with
NELSON
1018
20
IS
+
75
+
;
U)
a 4 C (0 -5
50 -4 10
-I-
-5 a
25 5
FIG. 3. Absorption of vitamin C as total amount and as percentage absorption in the 30-cm study segment from orange juice (hatched bars) and synthetic (clear bars) solutions. Means ± SEM indicated. Neither parameter shows statistically significant (P 0.05) difference. I
00
0
4
8C
Previous investigations have shown that glucose can enhance folate absorption (20). The absorption of water-soluble vitamins, ascorbic acid and folic acid did not differ in the two solutions despite a large difference in water absorption; this result, however, may be explained as follows. The orange juice solution may contain a “binder” which prevents augmentation of absorption of folate and vitamin C, despite enhanced water transport. Thus, if glucose were present in the synthetic mixture, the absorption of the waten-soluble vitamins might be increased over that from the orange juice mixture. The lack of correlation of synthetic folate absorption with water absorption contrasts with the positive correlation of water and vitamin C absorption. Differences in mode of transport, i.e., active transport versus passive diffusion, might be one explanation. Currently, studies are in progress to evaluate these possibilities. It is the conclusion of this study that there
is avid
IS
C
ET AL.
0
U
FIG. 4. Absorption of folic acid as total amount and as percentage absorption in the 30-cm study segment from orange juice (hatched bars) and synthetic (clear bars) solutions. Means ± SEM indicated. Neither parameter shows statistically significant (P 0.05) difference.
absorption
of vitamin
C and
folic acid
in the proximal jejunum from a synthetic and a food (citrus) source. These sources did not differ significantly in the availability of these nutrients. We conclude, also, that the method of triple lumen intestinal perfusion can quantitate absorption of water-soluble vitamins from food sources in human volunteers and that such data can be used to relate absorption of nutrients from a food product to the absorption of their synthetic counterparts. The method, then, is useful as applied to the problems of comparative bioavailability in human nutrition. The authors express appreciation Green, Mrs. Wanda Durrance, Mrs. and Mrs. Muriel Hogan for their
to Miss Bonnye Eugenia Ahmed, expert technical
assistance.
the data of Butterworth et al. (19), but not with that of Gerson et al. (20). The observed results of augmented water absorption with the orange juice mixture are consistent with previous data that glucose in concentrations of 14 mrs (240 mg/l00 ml) or greater promotes absorption of Nat, C1 and water (21). Maximum rates of water absorption are obtained from isotonic solutions containing approximately 56 mM (or 1%) glucose. The orangejuice solution used in this study contained 1.2% glucose; therefore, enhanced water absorption was expected.
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D. S.
G. E., D. A.
WHALEN,
BIOAVAILABILITY
Lancet
BAUGH
AND
AND
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Absorption
A. M.
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N. Folic
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