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Ascorbic Acids enhancing effect on the absorption of Non-Heme Iron

By
Kerianne Hofsiss



For
Dr. William R. Proulx, RD
Associate Professor of Nutrition & Dietetics



In partial fulfillment for the requirements of
NUTR342 Advanced Nutrition II

April 15, 2014


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TABLE OF CONTENTS
I. INTRODUCTION... 3

II. REVIEW OF LITERATURE.. 5

A. Absorption of Non-Heme Iron ... 5
B. Vitamin C enhance None Heme iron absorption.. 7
Ascorbic Acid Reduction of Ferric to Ferrous Iron 7
Ascorbic Acids Chelation Ability......................... 8
Hepcidin Expression Suppressed by Ascorbate .... 10

III. CONCLUSION 11

IV. REFERENCE LIST . 12

















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INTRODUCTION
Iron is a trace mineral that is essential for maintaining normal physiological functions. It
is an essential part of numerous enzymatic functions along with the production of red blood cells.
(1) Through the diet iron is either in the form of heme iron or as inorganic, non-heme iron.
Heme iron is a product of hemeprotein digestion, such as hemoglobin and myoglobin, within
animals and is therefore derived from dietary animal protein consumption (2). This iron is
absorbed much more efficiently than irons organic form non-heme iron. Non-heme iron is found
in greater abundance within plant based products rather than animal proteins and is therefore
often accompanied by inhibitory dietary factors that decrease its absorption. Non-heme iron
accounts for nearly 90% of our dietary iron consumption, and as a result much of the dietary iron
consumed is not efficiently absorbed (3). As a result, amongst the entire global population the
most common nutrient deficiency is iron. Iron deficiency anemia affects nearly 20% of the
population worldwide (4). For this reason extensive research has been done to determine the
mechanisms responsible for and regulatory functions of its absorption. Heme and non-heme iron
follow two different paths after ingestion. Since non-heme iron is the most common dietary
form of iron, it has attracted the most interest. Non-heme iron is directly influenced by other
dietary components. There are many known inhibitors of non-heme transport and just a few
known enhancers. Due to prevelance of anemia across the world, the enhancing ability of
ascorbic acid on non-heme iron absorption has raised much attention. Numerous research studies
have been conducted in order to test ascorbic acids enhancing ability and determine by what
mechanism its absorption is being enhanced. Determining the mechanisms by which ascorbic
acid is able to enhance non-heme iron absorption will result in a better understanding of exactly

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how ascorbic acid should be supplemented in the diet in order to achieve its maximum potential
of enhancement.





































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Review of Literature


Absorption of Non-Heme Iron

The degree of bioavailability of heme vs. non-heme iron is the main determinant of how
efficiently they are absorbed. Heme iron has a high bioavailability due to the mechanism
responsible for its absorption as well as the absence of inhibitors. This exact mechanism
responsible for heme iron absorption is still unclear. The latest research has indicated that there
is a receptor that is specific to heme iron that initiates endocytosis without need for special
carriers or transporter; which are required for non-heme endocytosis. (3). Non-heme iron has
low bioavailability in alkaline environments and therefore undergoes reduced absorption within
the lumen. Non-heme iron has found to be more readily oxidized to its insoluble form when
transported into the lumen. Non-heme iron happens to also be found in foods that contain
inhibitors such as phytates. Inhibitors form chelates with the iron making them insoluble and
thus reduce absorption (5). For iron to be absorbed properly receptor mediated endocytosis is
necessary. (6) After iron uptake into cell has occurred it is either stored as ferritin or transported
by the protein transferrin through plasma and received by erthyrpoiteic cells (7). The binding of
iron to transferrin for transport is necessary to prevent iron from damaging the body due to its
high redox potential. Iron bound to transferrin binds with TfR1 receptor on the cell membrane
and forms a complex. This complex can than undergo endocytosis into the cell (8). Before iron
can be stored or transferred it must first be absorbed and transported through bothe the apical and
basolateral membrane. 90% of iron found in the lumen is as an insoluble ferric form and
requires reduction before pick up by Divalent Mineral transporter 1, DMT1, for transport across
the apical membrane. DMT1 is otherwise known as Nramp 2, natural resistance associated
macrophage protein 2. (9). Along with the reduction of Ferric to Ferrous, DMT1 also requires the

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utilization of a proton gradient to release the iron from the endosomal compartment and into the
cytoplasm (6). In order for Fe3+ to be reduced it must undergo an enzymatic reduction by the
brush border ferriductases.(10). Dcytb, Duodenal cytochrome b, is the enzyme that catalyzes the
reduction of Fe3+. DMT-1 is can only transport Fe2+ into the cell. (11). A number of events can
occur that result in iron not being picked up by DMT1 and therefore not properly absorbed. Non-
heme is influenced greatly by meal composition and is more likely to form insoluble chelates or
become oxidized within in the lumen. For this reason non-heme iron also uses another
mechanism of absorption; the -integrin-mobilferrin pathway. Also called the paraferritin
complex, it consists of a three integrin, mobilferrin and a flavin monooxygenase (12). This
mechanism begins in the stomach where large glycoproteins called mucins bind with iron and are
then transported into the lumen as an iron-mucin complex. The iron-mucin complex prevents
polymerisations of iron hydroxide while also keeping iron soluble within the lumen, allowing
uptake by -integrin. (13). Mobilferrin, a 56 KD iron binding protein found within the
cytoplasm, takes up iron and exports it from the endosomal membrane compartment and
transports it into the cytoplasm (14). Regulation of this pathway is dependent upon the amount
of available binding sites on mobilferrin. For non-heme iron absorption to occur, the process
requires many other elements in order to keep iron soluble and ready for uptake. Non-heme
irons low bio availability is due to its state of insolubility; resulting from presence of dietary
inhibitors and oxidation within the lumen. Dietary enhancers, such as ascorbic acid, can
overcome inhibitors and reduce oxidation. Ascorbic acid has gained much attention for its
ability to increase the absorption rate and is a potential solution for the correction as well as
prevention of anemia.


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Vitamin C Enhances Non-Heme Iron absorption
There are many known dietary inhibitors of non-heme iron. Foods containing phytates,
tannins, polyphenols and calcium all have been identified as inhibitors. Ascorbic acid acts
opposite of these and has been shown to enhance the absorption of non-heme iron. Kaur and
Sangha recently found that amongst ninety girls between the ages of 16 and 18, all of whom
were anemic, ascorbic acid enhanced there iron absorption. Three groups of thirty girls were
given Iron-Folic supplements over three months. The control group just took the supplement, the
second group was given an additional vitamin C supplement, and the third group was given a
natural source of vitamin C, lemon water, with the iron-folic tablet. The two groups receiving a
supplemental form of ascorbic acid saw both their red blood cell level counts return to normal
along with hemoglobin levels (15). Ascorbic acid was able to successfully enhance absorption
of the iron supplement. There are numerous proposed mechanisms depicting the exact role
ascorbic acid facilitates in the enhancement of non-heme iron absorption.

Ascorbic Acid Reduction of Ferric to Ferrous Iron
Irons bio-availability is dependent upon its specific oxidation state. Iron has an
extremely sensitive redox potential of switching between ferric and ferrous. Non-heme must be
in ferrous form for absorption. However, because of the sensitivity of irons oxidation states, free
iron is not found often due to its potential to form free radicals and therefore must be carried by
proteins or chelated so that it remains as ferric, its unreactive form. In this oxidized state, as
ferric iron, it is incapable of undergoing endocytosis and cannot enter enterocytes. Ascorbic
acids antioxidant properties can resolve this issue by acting as a reducing agent to ferric iron
(Fe3+). Ascorbic acid maintains iron in ferrous form and therefore can be a replacement for the

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ferrireductase normally responsible for this action, Dcytb. (16). Ascorbic acid supplies electrons
and makes ferrous iron available for transport by DMT1. Xiamoin Lou used mice with low
ascorbate levels to prove that it could serve as a replacement. The mice showed an increased
expression of Dcytb. Once ascorbate was administered to the mice, their levels of Dcytb
decreased concluding that it was no longer needed as an electron donor in the presence of
ascorbate. (11). Evidence had also been previously discovered supporting that both the rate of
iron uptake as well as reduction of Fe3+ were increased when ascorbate was present. Thumser et
al. used cultured Caco-2 cells to test this. It was found that increased iron uptake occurred as a
result of an ascorbate-iron concentration dependent mechanism. Ascorbate levels administered
at the time of iron had a direct correlation with one another and as the ratio of ascorbate to iron
increased so did reduction and uptake of iron (1).

Ascorbic Acids Chelation Ability
Due to non-heme irons potential to form insoluble precipitates, further preventative
action is required. Reduction is only an option if iron has not already formed a complex
rendering it insoluble. Chelation before entering or while within the lumen can result in
inhibition or enhancement of absorption. One of the biggest examples of iron chelation is the
mechanism by which transferrin functions. It forms a chelate with iron to keep it in an
unreactive state while being transported, a change in pH is required to break up this ligand.
However, non-heme iron is highly susceptible to other chelators that inhibit absorption and form
insoluble ligands before it has gotten into the cell. Non-heme is derived from the diet from foods
that are rich in inhibitors such as tannins and phytates (13). The composition of the meal that
contains the non-heme iron along with additional foods taken along with it can either enhance or

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inhibit its absorption. Ascorbic acid can prevent the formation of these insoluble complexes and
is known as a dietary enhancer of non-heme iron absorption (17). Ascorbic acid was found to be
able to form a chelate with iron within in acid environment and stay a soluble complex when
transported into a more alkaline environment such as the duodenum. By forming this complex
ascorbic acid is able to counteract any ligands that may inhibit its absorption. Many iron
fortified foods contain ascorbic acid because of its ability to reduce ferric iron but it ability to
counteract inhibitors has also gained much interest. Research has shown that not only does
ascorbic acid prevent the formation of insoluble ligands, but that its effect on absorption rate
increases even further by the presence of inhibitors. When measuring the rate of absorption of
iron from meats and again from foods containing phytates and tannins, it was found that ascorbic
acids presence caused the rate of absorption to be higher in foods containing inhibtors than in
foods without them such as meats. Two meals were given to subjects, one with iron content
being from meat and the other iron from plants containing phytate. Each meal had 100mg
ascorbic acid added to them and it was found that the meal containing phytates iron absorption
increased by 4.15 while the meal without inhibitors only increased by 1.6 (18). Trinidad et al
conducted a different research study using 19 children. Over two days the children were given
two different iron-fortified drinks. One containing inhibitors and the other without. Iron
isotopes were used as markers of iron absorption. The beverages containing fiber and phytic
acid has more pronounced increases as the ratio of ascorbic to iron increased It was found that
when 40mg ascorbic acid were added to these beverages an increase of 1.5% iron absorption
occurred in both. In additions of 60mg iron absorption increased to 6% in the inhibitor containg
beverage and only by 4% in the other. A higher ratio of ascorbic to iron is needed to both
overcome inhibitors and enhance absorption even further than if no inhibitors were present(19).

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Zariwala et al. used ascorbyl palmitate nanocarriers to measure the absorption rate of iron when
in the presence of ascorbic acid. It was observed that ascorbic acid increased the rate of
absorption in adult women along with infants being fed fortified beverages/food. A molar ratio
of 2:1 is required in foods not containing inhibitors and foods containing inhibitors 4:1 ratio of
ascorbic to iron resulted in a significant increase in rate of absorption (4). The previously
mentioned study by Thumser et al. using Caco-2 cells also proposed that ascorbic acid acts as a
chelator by forming an insoluble complex with ascorbic acid. By measuring the kinetics of iron
uptake with and without ascorbic acid presence and plotting them they found ascorbate uptake to
be saturable. This suggests that the complex formed between iron and ascorbate can directly
enter into the cell as a single soluble complex (1).

Hepcidin Expression Suppressed by Ascorbate
Hepcidin is a peptide originating from the liver and is closely involved in the absorption
of both recycled and de novo iron along with regulating the transport of iron by ferroportin (2).
Ferroportin is the protein responsible for the release of iron into circulation. During times of iron
overload hepcidin expression is increased and during low levels of iron such as anemia it is
suppressed. Chui et al. recently attempted to find, if any, the correlation between hepcidin and
ascorbic acid. Using HepG2 cells, they inoculated a medium and added to it an additional 50 to
100g/mL of ascorbic acid. After 6 hours they measured the amount of hepcidin produced by
the HepG2 cells. It was found that with the addition of ascorbic acid there was a decrease in the
expression of hepcidin. Lower hepicidin suppression results in the more efficient use of recycled
iron. Therefore Chiu et al. found that ascorbic acid may also be used in iron deficient individual
to not just enhance iron absorption but by increasing the more efficient utilization of stored iron,
ferritin (9)

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CONCLUSION

Ascorbic acid is one of the most well-known enhancers of the abosprtion of non-heme
iron. Iron deficiency is responsible for majority of the cases of anemia found throughout the
world. For this reason, extensive research has been conducted to find ways to increase iron
absorption within populations. Non-heme iron, being the biggest dietary contributor form of iron
has received the most attention. Throughout the review of literature, the studies and conclusions
made all were unanimous in the enhancing effect of ascorbic acid on the efficiency of non-heme
absorption. The evidence has shown the most likely mechanisms responsible for this are in
ascorbic acids ability to reduce ferric iron to its soluble form ferrous and its ability to form a
chelate with iron over inhibitors. Ascorbic acid can replace the ferrireductase, Dcytb and use its
antioxidant properties to donate electrons to ferric iron to convert it into a soluble, absorbable
form. Chelation of iron with ascorbic acid, another valid mechanism responsible for its
enhancing ability, prevents certain inhibitors commonly found in foods containing iron such as
phytates and tannins to form a chelate. The ascorbate- iron complex has the ability of
overcoming the inhibitory effect of phytates and tannins on non-heme absorption. The proposed
mechanism of ascorbates suppressing effect on hepcidin expression is a feasible proposition for
another mechanisms responsible but needs to be researched more to be further validated. Overall
ascorbic acids enhancing effect on absorption of non-heme iron has been successfully shown
through numerous research studies. Ascorbic acid is accepted as a viable supplement when
given with meals containing non-heme iron in order to correct and prevent anemia caused by
iron deficiency.


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REFERENCE LIST

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4. Zariwala M. Gulrez, Farnaud Sebastien, Merhcant Zahra, et al. Ascorbyl palmitat/DSPE-
PEG nanocarriers for oral iron delivery: Preparation, characterization and in vitro
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Receptor in HepG2 cells. Journal of Renal Nutrition. 2012;22:373-376
10. Kim Young Eun, Ham SooKyung, Bradke Daniel et al. Ascorbic Acid Offsets the Inhibitory
Effect of Bioactive Dietary Polyphenolic Compounds on Transepithelial Iron Transport in
Caco-2 Intestinal Cells. The Journal of Nutrition. 2010;141:828-834
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Uptake. Pharmocology & Toxicology. 2002;91:97-102

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14. Conrad M, E. Umbreit J, N. Moore E, G. Iron absorption and Transport. American Journal of
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