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Institute of Food Research, Norwich Research Park, Colney, Norwich, United Kingdom
2To whom correspondence should be addressed. E-mail: sue.fairweather-tait{at}bbsrc.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONAvailable standardized and...Isotopic labeling of iron...Hemoglobin incorporationFecal monitoringWhole-body countingPlasma appearance/disappearanceCaco-2 cell systemsNormalization of resultsREFERENCES |
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KEY WORDS: • bioavailability • supplements • iron
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONAvailable standardized and...Isotopic labeling of iron...Hemoglobin incorporationFecal monitoringWhole-body countingPlasma appearance/disappearanceCaco-2 cell systemsNormalization of resultsREFERENCES |
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, van Dokkum et al. 1996
, Wienk et al. 1999
). The aim of the present article is to focus on
methods that can be used to assess the bioavailability of iron
supplements, not dietary iron. However, the bioavailability of any iron
supplement taken with food(s) or beverage(s) will be modified by the
presence of a wide range of dietary inhibitors and enhancers.
Similarly, physiological factors that determine the efficiency of
dietary iron absorption will also influence the absorption of
supplemental iron (Fairweather-Tait 1998
).
Dietary iron is found in two predominant forms, heme and nonheme, and
these are absorbed by separate pathways, heme as the intact moiety and
nonheme iron from the common iron pool within the gut (Hallberg 1981
). Because heme iron is rarely, if ever, used as an iron
supplement per se, the methods discussed in this article relate
specifically to nonheme iron supplements.
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Available standardized and validated methods |
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TOPABSTRACTINTRODUCTIONAvailable standardized and...Isotopic labeling of iron...Hemoglobin incorporationFecal monitoringWhole-body countingPlasma appearance/disappearanceCaco-2 cell systemsNormalization of resultsREFERENCES |
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80–90% of absorbed iron is
used for the synthesis of hemoglobin in reticulocytes, bioavailability
can be measured as hemoglobin incorporation using isotopes. However,
because there are no major excretory pathways for iron, various
measures of absorption are also used to predict bioavailability.
Early studies made use of animal models, particularly the rat, for
assessing the bioavailability of different forms of iron. However,
there are some major discrepancies between results from studies in
rodents and man, and it is now generally accepted that rats cannot be
used to assess the quantitative importance of dietary factors in human
iron nutrition (Reddy and Cook 1991
). Therefore, animal
models are not recommended for studying the bioavailability of iron
supplements.
Techniques currently used to assess iron bioavailability are
summarized in Table 1
. Most involve radio- or stable isotope labels to monitor the absorption
and/or subsequent metabolism of different forms of iron, but it is also
possible to assess bioavailability by measuring the rate of hemoglobin
repletion in anemic individuals. However, this requires recruitment of
volunteers who are already anemic or who are made anemic by
venesection, which may create logistical and/or ethical problems.
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Isotopic labeling of iron supplements |
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TOPABSTRACTINTRODUCTIONAvailable standardized and...Isotopic labeling of iron...Hemoglobin incorporationFecal monitoringWhole-body countingPlasma appearance/disappearanceCaco-2 cell systemsNormalization of resultsREFERENCES |
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. Some are slow release preparations, designed to reduce unpleasant
gastrointestinal side effects. Efficacy is clearly improved when
bioavailability is maximized, but for people who are not iron deficient
and, hence, will not absorb iron efficiently, the advisability of
ingesting high doses of oral iron has been questioned, particularly in
relation to increased risk of bowel cancer (Wurze-mann et al. 1996
). The mechanism is not understood but it may relate to the
generation of free radicals in feces, as demonstrated with oral ferrous
sulfate supplements in iron-replete individuals (Lund et al. 1999
). Thus, bioavailability is a key determinant of both
efficacy and safety.
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-Fe2-O3) and magnetite
(Fe3O4) and metallic iron.
There are two radioisotopes of iron, 59Fe and
55Fe, both of which have been used extensively
for human iron studies (Hallberg 1981
). These are
generally supplied as high specific activity ferric chloride solution.
More recently, attention has focused on developing stable isotope
techniques for studying iron bioavailability because of ethical
concerns about exposure to ionizing radiation. Iron has four stable
isotopes, three of which (54Fe,
57Fe and 58Fe) have a low
enough natural abundance for iron bioavailability studies (5.8, 2.2 and
0.28%, respectively).
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Hemoglobin incorporation |
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TOPABSTRACTINTRODUCTIONAvailable standardized and...Isotopic labeling of iron...Hemoglobin incorporationFecal monitoringWhole-body countingPlasma appearance/disappearanceCaco-2 cell systemsNormalization of resultsREFERENCES |
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). A comparison can be made between two different sources
of iron, labeled with 55Fe or
59Fe, or between the test compound and a
well-absorbed reference dose comprised of 3 mg of iron as ferrous
sulfate plus 30 mg of ascorbic acid (Hallberg et al. 1997
). Sensitive sample preparation and detection methods have
been developed that require the administration of levels of
radioactivity as low as 37 kBq of 59Fe or 111 kBq
of 55Fe. The percentage of iron absorption is
calculated from estimates of blood volume (based on a height and weight
nomogram) and an assumed red cell incorporation of absorbed iron of 80
or 100% [with serum ferritin concentration > 15 or < 15
µg/L, respectively (Cook et al. 1991
)].
An alternative, but rarely used, dual isotope method for obtaining
absolute instead of relative values for iron absorption can be used
(Saylor and Finch 1953
) that avoids the need for making
assumptions about percent incorporation of absorbed iron into red blood
cells. In this method, 59Fe is given orally at
the same time that 55Fe, bound to plasma, is
injected intravenously. Absorption is calculated by relating the ratio
of the two isotopes in the red cells to the ratio of administered
isotopes, and whole-body 59Fe counting can be
used for verification. Stable isotopes have also been used to measure
absorption in women from an oral dose of 5 mg of
57Fe and an intravenous dose of 250 µg of
58Fe (Barrett et al. 1994
). In
principle, the method could be used for studying more than one iron
compound if mixtures of radio- and stable isotopes are used.
When radioisotope administration is not permitted, hemoglobin
incorporation of stable isotopes can be measured to assess iron
bioavailability (van Dokkum et al. 1996
). Unlike the
situation with radioisotopes, where background activity is not a
problem, relatively large doses of stable isotopes have to be given to
produce measurable enrichment of red blood cells. This creates labeling
problems for food iron, where intakes of iron are relatively low, and
necessitates multiple dosing protocols. Thus, early applications of
stable isotope methodology were in infants (Fairweather-Tait et al. 1995
) whose blood volume is smaller than that of adults.
However, the constraints concerning bioavailability measurements of
iron supplements relate to the cost of isotope and labeling of the
iron, not to dose per se. Increased sensitivity may be achieved by
separating the young erythroid cells from blood for the determination
of isotopic enrichment, and this will reduce the oral dose of isotope
by up to one third (van den Heuvel et al. 1998
).
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Fecal monitoring |
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TOPABSTRACTINTRODUCTIONAvailable standardized and...Isotopic labeling of iron...Hemoglobin incorporationFecal monitoringWhole-body countingPlasma appearance/disappearanceCaco-2 cell systemsNormalization of resultsREFERENCES |
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The main problem with fecal monitoring is incomplete fecal collection,
particularly in individuals with long transit times. Short collection
periods may lead to an overestimate of absorption due to incomplete
recovery of unabsorbed isotope from luminal contents and/or incomplete
collection of mucosal cells that contain isotope that was not
transferred to the systemic circulation. A number of markers have been
used to test for completeness of fecal collection, the most recent
being the rare earth elements (Fairweather-Tait et al. 1997
).
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Whole-body counting |
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TOPABSTRACTINTRODUCTIONAvailable standardized and...Isotopic labeling of iron...Hemoglobin incorporationFecal monitoringWhole-body countingPlasma appearance/disappearanceCaco-2 cell systemsNormalization of resultsREFERENCES |
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).
The supplement must be labeled with the gamma-isotope,
59Fe. An initial baseline count is made to
measure background activity (from 40K and any
residual radioactivity from previous studies involving radioisotopes)
and the volunteer is given 59Fe-labeled iron
compound and recounted 1–5 h and 10–14 d later. The difference is
calculated as absorbed isotope, once corrections have been made for
isotope decay and geometry. |
Plasma appearance/disappearance |
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TOPABSTRACTINTRODUCTIONAvailable standardized and...Isotopic labeling of iron...Hemoglobin incorporationFecal monitoringWhole-body countingPlasma appearance/disappearanceCaco-2 cell systemsNormalization of resultsREFERENCES |
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). To quantify iron absorption using
plasma appearance data, information on the rate of removal of iron from
the plasma is required; this can be obtained by giving an intravenous
dose of a second iron isotope at the same time as the oral dose and
monitoring its concentration in plasma. Plasma concentrations of the
isotopes are calculated using deconvolution analysis to obtain the area
under the curve. In practice, it is often not possible to take blood
samples until all of the isotopes have disappeared from the plasma;
therefore, a simulation has to be performed from the last sample point.
This is more straightforward for the intravenous dose (which follows an
exponential decay) than for the oral dose. To simulate beyond the last
point of the oral data, sampling must be taken beyond the time required
for absorption to be completed and the subsequent appearance of oral
isotope is assumed to follow the same form of exponential decay as the
intravenous isotope.
A simpler method for assessing bioavailability that does not require
isotopic labeling can be used for large (25–100 mg) doses of iron. The
serum iron increase 4–6 h postingestion is significantly correlated
with absorption of 59Fe measured by
whole-body counting (Ekenved et al. 1976
). It is
possible to use this technique to make a qualitative assessment of the
bioavailability of different iron supplements.
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Caco-2 cell systems |
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TOPABSTRACTINTRODUCTIONAvailable standardized and...Isotopic labeling of iron...Hemoglobin incorporationFecal monitoringWhole-body countingPlasma appearance/disappearanceCaco-2 cell systemsNormalization of resultsREFERENCES |
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) and meal digests (Glahn et al. 1996
).
Caco-2 TC7 cells have recently been shown to express Nramp2 (a divalent
cation transporter) in the apical membrane, with a substrate preference
for iron and transport occurring by a proton-dependent mechanism
(Tandy et al. 2000
). These results indicate that this in
vitro system could provide a rapid screening technique to measure the
maximum potential bioavailability of iron supplements, provided the
iron can be labeled isotopically. |
Normalization of results |
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TOPABSTRACTINTRODUCTIONAvailable standardized and...Isotopic labeling of iron...Hemoglobin incorporationFecal monitoringWhole-body countingPlasma appearance/disappearanceCaco-2 cell systemsNormalization of resultsREFERENCES |
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).
Multiple dosing protocols can reduce day-to-day fluctuations in
efficiency of absorption, but this approach is not always possible.
Interindividual variation is primarily due to differences in body iron
stores and, hence, physiological requirements for iron (Hallberg et al. 1998
). Two main approaches have been proposed to
normalize the differences.
).
) using the equation:
 |
Recently, Hallberg et al. (1998)
examined the
relationship between iron stores and iron absorption in relation to
dietary requirements for iron. They have developed a new equation
relating serum ferritin and iron stores, which may affect the
normalization procedure. They also emphasize the inertia in changes of
iron stores by presenting a series of graphs illustrating the rate of
increase of iron stores in individuals with different iron
requirements. The latter clearly demonstrates that it takes years, not
months, for iron stores to reach a plateau, regardless of dietary iron
bioavailability. Thus, long-term endpoints are not appropriate for
studying the bioavailability of iron supplements.
The best method for assessing bioavailability of supplemental iron is hemoglobin incorporation, followed by fecal monitoring, using radioisotope or stable isotope labeling. Caco-2 cell systems can be used for rapid screening of labeled supplements to predict availability for absorption. If the compound cannot be labeled, then plasma appearance/disappearance of oral iron given together with an intravenous dose of iron isotope can be used to quantify absorption. With oral doses in excess of 25 mg, the 4- to 6-h plasma iron concentration can be taken as a qualitative assessment of iron bioavailability. For all methods, it is important to design a protocol that enables the results from individual studies to be normalized, for example, by comparing the supplement with an iron source of known bioavailability, e.g., 3 mg of ferrous sulfate plus 30 mg of ascorbic acid, or to estimate maximum potential bioavailability.
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONAvailable standardized and...Isotopic labeling of iron...Hemoglobin incorporationFecal monitoringWhole-body countingPlasma appearance/disappearanceCaco-2 cell systemsNormalization of resultsREFERENCES |
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Tandy S., Williams M., Leggett A., Lopez-Jimenez M., Dedes M., Ramesh B., Srai S. K., Sharp P. Nramp2 expression is associated with pH- dependent iron uptake across the apical membrane of human intestinal Caco-2 cells. J. Biol. Chem. 2000;275:1023-1029
20.
van den Heuvel E., Muys T., Pellegrom H., Bruyntjes J. P., van Dokkum W., Spanhaak S., Schaafsma G. A new method to measure iron absorption from the enrichment of 57Fe and 58Fe in young erythroid cells. Clin. Chem. 1998;44:649-654
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