A Porcine-Derived Heme Iron Powder Restores Hemoglobin in Anemic Rats

Abstract

Background/Objectives: Iron-fortified foods reduce the incidence of iron deficiency anemia. However, the nutritional efficacy of heme iron fortificants is unclear. Methods: In this study, we determined the hemoglobin regeneration efficiency (HRE) of a porcine-derived heme iron powder (HIP), treating anemic rats (hemoglobin (Hb) 3–6 g/dL) with 14-day repletion diets fortified with four different concentrations (12, 24, 36, or 48 mg iron/kg diets) of HIP or a control diet (“no added iron”); n = 9–12/group. Results: Our results demonstrate an inverse association between HRE and increasing dietary iron from the HIP. The HRE ratios of diets containing the HIP powder at 12, 24, 36, or 48 mg iron/kg were 0.508, 0.268, 0.273, and 0.223, respectively. Based on the mean final Hb values at 14 d, the HRE ratio of the 12 mg iron/kg diet was significantly higher (p ≤ 0.05) compared to the other HIP diet groups; however, only the HIP provided in the 36 and 48 mg iron/kg diets restored hemoglobin to high enough levels (mean Hb > 6 g/dL) to correct anemia. Conclusions: Our findings show that HIP at each of the concentrations tested increased Hb; moreover, when present at higher concentrations in the diet, the HIP is capable of restoring hemoglobin to resolve iron deficiency anemia.

Full text

Citation: Swain, J.H.; Glosser, L.D. A
Porcine-Derived Heme Iron Powder
Restores Hemoglobin in Anemic Rats.
Nutrients 2024,16, 4029. https://
doi.org/10.3390/nu16234029
Academic Editor: Zumin Shi
Received: 29 September 2024
Revised: 7 November 2024
Accepted: 22 November 2024
Published: 25 November 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Article
A Porcine-Derived Heme Iron Powder Restores Hemoglobin in
Anemic Rats
James H. Swain 1, * and Logan D. Glosser 2
1Department of Nutrition, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue,
Cleveland, OH 44106, USA
2School of Medicine, Emory University, 100 Woodruff Circle, Atlanta, GA 30322, USA
*Correspondence: james.swain@case.edu; Tel.: +1-216-368-8554
Abstract: Background/Objectives: Iron-fortified foods reduce the incidence of iron deficiency anemia.
However, the nutritional efficacy of heme iron fortificants is unclear. Methods: In this study, we
determined the hemoglobin regeneration efficiency (HRE) of a porcine-derived heme iron powder
(HIP), treating anemic rats (hemoglobin (Hb) 3–6 g/dL) with 14-day repletion diets fortified with
four different concentrations (12, 24, 36, or 48 mg iron/kg diets) of HIP or a control diet (“no added
iron”); n= 9–12/group. Results: Our results demonstrate an inverse association between HRE and
increasing dietary iron from the HIP. The HRE ratios of diets containing the HIP powder at 12, 24,
36, or 48 mg iron/kg were 0.508, 0.268, 0.273, and 0.223, respectively. Based on the mean final Hb
values at 14 d, the HRE ratio of the 12 mg iron/kg diet was significantly higher (p
≤
0.05) compared
to the other HIP diet groups; however, only the HIP provided in the 36 and 48 mg iron/kg diets
restored hemoglobin to high enough levels (mean Hb > 6 g/dL) to correct anemia. Conclusions:
Our findings show that HIP at each of the concentrations tested increased Hb; moreover, when
present at higher concentrations in the diet, the HIP is capable of restoring hemoglobin to resolve
iron deficiency anemia.
Keywords: heme iron; iron absorption; hemoglobin regeneration efficiency; micronutrient; food
fortification
1. Introduction
Iron deficiency anemia (IDA) is the most prevalent micronutrient deficiency among
humans worldwide [
1
–
3
]. IDA results in significant reductions in work productivity and
adds billions of dollars to the cost of health care, burdening already stressed medical
systems [
4
]. To correct IDA, staple foods, especially cereal grain flours, are fortified with
different forms of iron, including elemental and heme sources [
5
–
8
]. However, the efficacy
of many types of iron fortificants is unclear [9–15].
Increasingly, there is interest in the use of bovine- and porcine-derived heme iron
powders (HIPs) to serve as fortificants in a variety of foods since iron in the heme form has
relatively good bioavailability and is less influenced by inhibitors of non-heme iron absorp-
tion, such as tannins, phytic acid, and calcium, which greatly reduce iron absorption [
16
–
19
].
Heme iron is also more highly absorbed and better tolerated (less gastrointestinal discom-
fort) when compared to non-heme iron powder intake; iron from HIPs also results in less
oxidative stress intraluminally [
9
,
11
,
13
,
19
,
20
]. HIPs are polypeptides that contain a soluble
heme moiety derived from the enzymatic digestion of bovine or porcine hemoglobin [
7
,
19
].
The bioavailability of iron from HIPs has been found to be 40–60% higher than iron from
non-heme elemental iron powders or iron salts, such as ferrous sulfate [
10
,
20
,
21
]. Although
heme iron intake contributes greatly to the overall iron absorption within a well-balanced,
omnivorous (meat-containing) diet, a better understanding of its absorption from commer-
cially prepared heme iron powder fortificants may assist in developing enhanced dietary
guidelines [21,22].
Nutrients 2024,16, 4029. https://doi.org/10.3390/nu16234029 https://www.mdpi.com/journal/nutrients

Nutrients 2024,16, 4029 2 of 10
Iron absorption, bioavailability, and the impact of different forms of dietary iron on
hemoglobin and iron status have been investigated in murine and avian models, as well as
in humans [
23
–
27
]. Among murine models, the rat hemoglobin repletion assay has been
shown to be an appropriate approach for studying iron intake and absorption, hemoglobin
response, and resolution of IDA using different forms of dietary iron [23,25–28].
The purpose of this study was to determine the hemoglobin regeneration efficiency
(HRE) of a heme iron powder (HIP) by determining the change in hemoglobin and
hemoglobin iron in anemic rats as they consumed graded (increased) quantities of HIP
during a 14-day iron repletion period. The aim was to better understand the absorption of
iron from the HIP and thereby help develop more specific dietary guidelines regarding the
use of HIPs as alternative food fortification approaches.
2. Materials and Methods
2.1. Heme Iron Powder
This study tested a heme iron powder (HIP) (Proliant, Inc., Ankeny, IA, USA/APC
Europe, S.A., Barcelona, Spain). A “no added iron” diet served as the control. The HIP was
spray-dried blood in powdered form (porcine origin), dark brown/black in color, similar to
that described previously [
7
]. The HIP used in this study had the following characteristics:
78.13% protein, 9.57% ash, 1.48% iron, and 5.6% humidity. The HIP was subjected to 80
◦
C
for at least 2 h during production. Upon receipt, the HIP was stored in a desiccator under
vacuum and refrigerated (3 ◦C) until use.
2.2. Study Design and Dietary Treatments
This study initially used 72 male weanling Sprague Dawley rats (Charles River/SASCO,
Wilmington, MA, USA). The rats were housed individually in stainless steel mesh wire-
bottom cages at 21
±
1
◦
C and provided with a 12-h light–dark cycle. After a 24-day
depletion period consuming an iron-deficient diet (1.6 mg iron/kg AIN-93G[M] diet;
approximately 1.4 mg iron/kg diet-analyzed iron content), anemic rats with hemoglobin
values between 3 and 6 g/dL (mean
±
SEM of 3.9
±
0.6 g/dL; range 3.1–5.8 g/dL) were
then randomly assigned to one of five different iron repletion-period diet groups, with
the blocking based on hemoglobin. The rats were then provided with repletion diets for
14 d, fortified with HIP or a control diet (“no added iron”); n= 9–12/group. All diets and
deionized, distilled water were provided ad libitum. The HRE ratio calculation accounts
for body weight and iron intake; therefore, during the repletion period, animal weight
and food consumption measurements were performed daily, including adjustments for
spilled food, to determine both daily and total iron intake. All animal procedures followed
the Institutional Animal Care and Use Committee (IACUC) procedures at Case Western
Reserve University (CWRU) in accordance with NIH guidelines.
The HIP was incorporated into diets modified to have a very low base iron content,
using vitamin-free casein (Harlan Teklad, Madison, WI, USA), a modified mineral mix
that omitted ferric citrate (Harlan Teklad), a high purity microcrystalline cellulose fiber
source (Alphacel™; ICN Biomedicals, Irvine, CA, USA), and reagent grade ingredients.
The diets were also phytate-free, with a neutral pH (7.0). The baseline-modified diet [
29
]
(AIN-93G[M]) composition, from which treatment (repletion-period) diets were prepared,
is shown in Table 1. Without added iron, the diet contained approximately 1.4 mg iron/kg
by analysis. To prepare repletion-period diets, the HIP was added, taking into account the
baseline amount (analyzed) already present in the control (“no added iron”) group.
Mixing of the HIP into repletion-period diets was performed as previously described [
5
].
The HIP contained 1.48% iron (w/w); the iron concentration present in the HIP was used to
determine the amounts added to the baseline diet to attain the desired iron concentrations in
treatment diets. Inductively coupled plasma optical emission spectroscopy (ICP-OES; Series
720/730; Agilent, Inc., Santa Clara, CA, USA) was used to confirm the iron concentration in
the HIP. Thereafter, prior to the repletion period, small portions of each repletion-period

Nutrients 2024,16, 4029 3 of 10
treatment diet and the control (“no added iron”) diet were also taken for analysis to confirm
the iron content as previously described [19].
Table 1. Treatment diets were prepared by adding iron (Fe) as heme iron powder (HIP) to the
following baseline diet composition 1.
Formula g/Kg
Corn Starch 397.5
Casein 2200
Maltodextrin 132
Sucrose 100
Soybean Oil 70
Cellulose, microcrystalline (Alphacel™) 50
Mineral Mix modified, no added iron (06053) 35
Vitamin Mix, AIN-93-VX (94047) 310
L-Cystine 3
Choline Bitartrate 2.5
TBHQ, antioxidant 40.014
Macronutrient % Dry Weight % Kcal
Protein 18.3 19.4
Carbohydrate 60.1 63.8
Fat 7 16.7
1
Ref: [
29
].
2
Alcohol-extracted, vitamin-free, casein.
3
Ascorbic acid at 200 mg/kg diet.
4
Tertiary-
butylhydroquinone. Catalog numbers are shown in parentheses for mineral and vitamin mixes at the time
of preparation. All diet ingredients were obtained from Harlan Teklad, Madison, WI, USA.
2.3. Hemoglobin and Hemoglobin Iron Determinations
Procedures for determining hemoglobin (Hb) and Hb iron, phlebotomy, and animal
sacrifice following anesthetization were conducted as previously described [
5
]. Briefly, the
following calculation was used to determined Hb iron:
Hb Fe (mg) = BW (kg) ×0.067 ×Grams Hb per mL ×3.35 mg Fe
It is important to note that the calculation assumes that the blood is 6.7% body weight
(BW; kg) and hemoglobin iron content is 3.35 mg/g [
8
,
26
]. Therefore, the Hb iron was
determined on the basis of 3.35 mg iron/g Hb and 0.075 L blood/kg body weight [23,28].
2.4. Hemoglobin Regeneration Efficiency
The hemoglobin (Hb) regeneration efficiency (HRE) of the HIP was determined as
described previously [
5
]. Briefly, HRE ratios were calculated using the analyzed value of
iron for each diet based on the following formula:
HRE ratio = [Final Hb Fe (mg) −Initial Hb Fe (mg)]/Fe intake (mg total consumed; analyzed diet value)
2.5. Statistical Analyses
A preliminary power analysis was conducted based on previously published
data [5,13,23]
. Statistical analyses of hematological indices and hemoglobin (Hb) repletion
data were performed as described previously [
5
,
23
,
28
,
30
–
32
]. The control diet (“no added
iron”) group served as a point of reference for comparing the Hb increase from the baseline
diet. Differences between the diet group mean values, including for HRE, were tested using
Tukey’s multiple comparison test and Duncan post-hoc testing using the statistical package
SAS (SAS Version 10.2, SAS Institute, Cary, NC, USA). Data illustrations were performed
using GraphPad Prism (Software version 10.2; GraphPad, Boston, MA, USA). Values were
expressed as mean values ±SEM. Significance was set at p≤0.05.

Nutrients 2024,16, 4029 4 of 10
3. Results
3.1. Hemoglobin and Hemoglobin Iron Change
The hematological values of anemic rats fed the control diet (“no added iron”) or on
the 12, 24, 36, and 48 mg iron/kg diets, with iron added in the form of heme iron powder
(HIP), are shown in Table 2. Food intake and weight gain were positively associated with
increasing dietary iron in all the treatment groups (Table 2). Iron intake (mg/day) was
positively associated with dietary iron concentration (Figure S1). Hemoglobin changes
and hemoglobin iron (Fe) gain in anemic rats fed HIP were positively associated with iron
concentration in the diet (Figure 1A,B).
Table 2. Food and iron (Fe) intake, growth, hemoglobin Fe change, and hemoglobin regeneration
efficiency (HRE) in anemic rats fed graded quantities of the heme iron powder (HIP) for a 14-day
repletion period 1,2.
Control
(“No Added Iron”)
Heme Iron Powder (HIP)
Diet Code C HIP-1 HIP-2 HIP-3 HIP-4
Diet Fe (mg/kg)
Calculated 1.6 12 24 36 48
(Analyzed) (1.4) (11.6) (25.3) (34.2) (47.1)
Food intake (g/day) 11.7 ±0.59 d13.2 ±0.64 c13.9 ±0.67 bc 15.1 ±0.85 ab 16.3 ±0.90 a
Fe intake (mg/day) 0.016 ±7−4 e 0.153 ±0.02 d0.352 ±0.06 c0.516 ±0.06 b0.768 ±0.09 a
Body weight (g)
Initial 83.9 ±3.8 a84.1 ±3.6 a83.6 ±3.5 a83.2 ±3.8 a85.5 ±3.7 a
(Gain) (15.2 ±0.9 c)(53.8 ±2.9 b) (54.9 ±3.0 ab) (56.3 ±3.4 ab)(59.4 ±3.5 a)
Hemoglobin (g/dL)
Initial 4.63 ±0.5 a4.82 ±0.7 a4.73 ±0.5 a4.79 ±0.6 a4.85 ±0.9 a
(Gain) (−0.42 ±0.04 e)(0.63 ±0.1 d)(1.16 ±0.3 c)(2.56 ±0.3 b)(3.28 ±0.4 a)
Final 4.21 ±0.4 c5.45 ±0.7 b5.89 ±0.9 b7.35 ±1.1 a8.13 ±1.3 a
Hemoglobin Fe 3
Gain (mg) 0.064 ±8−3 e 0.777 ±0.06 d0.944 ±0.07 c1.407 ±0.09 b1.713 ±0.09 a
HRE 4,5 - 0.508 ±0.06 a0.268 ±0.03 b0.273 ±0.04 bc 0.223 ±0.02 c
1
Values are mean values
±
SEM (n= 9–12/group).
2
Different letters (a–e) are used to denote significant
differences (p
≤
0.05) from higher to lower mean values within a row.
3
Hb Fe (mg) = BW (body weight; kg)
×
0.067
×
Grams Hb per mL
×
3.35 mg Fe.
4
HRE ratio = [Final Hb Fe (mg)
−
Initial Hb Fe (mg)]/Fe intake (mg
total consumed).
5
A dash mark in the “No Added Iron” column for HRE indicates it is not applicable since there
was no Hb increase in the control.
3.2. Hemoglobin Regeneration Efficiency of Heme Iron Powder
Hemoglobin regeneration efficiency (HRE) ratios of diets containing HIP are also
shown in Table 2. The HRE ratio calculation accounts for body weight and iron intake.
The HRE ratios of HIP diets containing 12, 24, 36, and 48 mg iron/kg expressed as mean
values ±SEM
were 0.508
±
0.06, 0.268
±
0.03, 0.273
±
0.04, and 0.223
±
0.03, respectively.
The comparative HRE ratios of the HIP at each level of dietary iron are shown in Figure 2.

Nutrients 2024,16, 4029 5 of 10
Nutrients 2024, 16, x FOR PEER REVIEW 5 of 10
Figure 1. (A) Hemoglobin (Hb) gain (g/dL). (B) Hemoglobin iron (Fe) gain in anemic rats fed 12, 24,
36, and 48 mg iron/kg diets in the form of heme iron powder for a 14-day repletion period. Values
are mean values ± SEM (n = 9–12/group). Different leers (a–d) are used to denote significant differ-
ences (p ≤ 0.05) from higher to lower hemoglobin and hemoglobin Fe gain. Hb Fe (mg) = BW (body
weight; kg) × 0.067 × Grams Hb per mL × 3.35 mg Fe.
3.2. Hemoglobin Regeneration Efficiency of Heme Iron Powder
Hemoglobin regeneration efficiency (HRE) ratios of diets containing HIP are also
shown in Table 2. The HRE ratio calculation accounts for body weight and iron intake.
The HRE ratios of HIP diets containing 12, 24, 36, and 48 mg iron/kg expressed as mean
values ± SEM were 0.508 ± 0.06, 0.268 ± 0.03, 0.273 ± 0.04, and 0.223 ± 0.03, respectively.
The comparative HRE ratios of the HIP at each level of dietary iron are shown in Figure
2.
Figure 1. (A) Hemoglobin (Hb) gain (g/dL). (B) Hemoglobin iron (Fe) gain in anemic rats fed 12, 24,
36, and 48 mg iron/kg diets in the form of heme iron powder for a 14-day repletion period. Values
are mean values
±
SEM (n= 9–12/group). Different letters (a–d) are used to denote significant
differences (p
≤
0.05) from higher to lower hemoglobin and hemoglobin Fe gain. Hb Fe (mg) = BW
(body weight; kg) ×0.067 ×Grams Hb per mL ×3.35 mg Fe.

Nutrients 2024,16, 4029 6 of 10
Nutrients 2024, 16, x FOR PEER REVIEW 6 of 10
Figure 2. Hemoglobin (Hb) regeneration efficiency (HRE) in anemic rats fed 12, 24, 36, and 48 mg
iron/kg diets in the form of heme iron powder for a 14-day repletion period. Values are mean values
± SEM (n = 9–12/group). Different leers (a–c) are used to denote significant differences (p ≤ 0.05)
from higher to lower HRE. HRE ratio = [Final Hb Fe (mg) − Initial Hb Fe (mg)]/Fe intake (mg total
consumed).
4. Discussion
Determining the hemoglobin regeneration efficiency using the rat hemoglobin reple-
tion assay has been identified by other research groups as a proficient and valid approach
to test the ability of different forms of dietary iron to resolve the anemia of iron deficiency
[23,24,28,30–35]. Findings from this study are valuable by demonstrating that iron from
this type of heme iron powder (HIP) increased hemoglobin at each of the concentrations
tested and that at higher concentrations in the diet (36 and 48 mg iron/kg), the HIP is
capable of restoring hemoglobin to resolve iron deficiency anemia (IDA). Our data are also
beneficial in illustrating the HRE ratios of the HIP at four different concentrations of iron
tested in a diet with minimal (negligible) background iron.
Findings from this study show that the hemoglobin change and hemoglobin iron gain
in anemic rats fed HIP were positively associated with iron concentration in the diet. Our
results are in agreement with the findings of other studies that have investigated the he-
matological effects of different forms of iron powders [26–28,30,34,35]. Overall, we found
that HRE was inversely associated with increasing dietary iron. Although the HRE ratio
of the diet containing the lowest iron concentration (12 mg iron/kg diet) was significantly
higher (p ≤ 0.05) than the other HIP diet groups based on the mean final hemoglobin at 14
d, only the HIP provided at the two greater concentrations of iron tested in this study (36
and 48 mg iron/kg diets) restored hemoglobin to adequate levels to correct anemia (Hb >
6 g/dL). A prior study on the effect of dietary iron levels on the efficiency of converting
non-heme iron into hemoglobin in anemic rats found that the efficiency of conversion of
dietary iron into hemoglobin iron was not significantly affected by the level of dietary iron
[35]. These results are in contrast to our results, which demonstrated that at lower levels,
HRE was significantly higher (p ≤ 0.05). Thus, our findings show a greater proportional
gain in hemoglobin with less heme iron consumed; as the amount of dietary iron from the
HIP increased, the proportional gain in hemoglobin was less. This result is similar to the
findings of previous studies that investigated the total percentage of dietary iron absorp-
tion [32,36].
Figure 2. Hemoglobin (Hb) regeneration efficiency (HRE) in anemic rats fed 12, 24, 36, and 48 mg
iron/kg diets in the form of heme iron powder for a 14-day repletion period. Values are mean
values ±SEM
(n= 9–12/group). Different letters (a–c) are used to denote significant differences
(
p≤0.05
) from higher to lower HRE. HRE ratio = [Final Hb Fe (mg)
−
Initial Hb Fe (mg)]/Fe intake
(mg total consumed).
4. Discussion
Determining the hemoglobin regeneration efficiency using the rat hemoglobin reple-
tion assay has been identified by other research groups as a proficient and valid approach
to test the ability of different forms of dietary iron to resolve the anemia of iron defi-
ciency [
23
,
24
,
28
,
30
–
35
]. Findings from this study are valuable by demonstrating that iron
from this type of heme iron powder (HIP) increased hemoglobin at each of the concentra-
tions tested and that at higher concentrations in the diet (36 and 48 mg iron/kg), the HIP is
capable of restoring hemoglobin to resolve iron deficiency anemia (IDA). Our data are also
beneficial in illustrating the HRE ratios of the HIP at four different concentrations of iron
tested in a diet with minimal (negligible) background iron.
Findings from this study show that the hemoglobin change and hemoglobin iron
gain in anemic rats fed HIP were positively associated with iron concentration in the diet.
Our results are in agreement with the findings of other studies that have investigated the
hematological effects of different forms of iron powders [
26
–
28
,
30
,
34
,
35
]. Overall, we found
that HRE was inversely associated with increasing dietary iron. Although the HRE ratio
of the diet containing the lowest iron concentration (12 mg iron/kg diet) was significantly
higher (p
≤
0.05) than the other HIP diet groups based on the mean final hemoglobin
at 14 d, only the HIP provided at the two greater concentrations of iron tested in this
study (36 and 48 mg iron/kg diets) restored hemoglobin to adequate levels to correct
anemia (Hb > 6 g/dL). A prior study on the effect of dietary iron levels on the efficiency
of converting non-heme iron into hemoglobin in anemic rats found that the efficiency of
conversion of dietary iron into hemoglobin iron was not significantly affected by the level
of dietary iron [
35
]. These results are in contrast to our results, which demonstrated that at
lower levels, HRE was significantly higher (p
≤
0.05). Thus, our findings show a greater
proportional gain in hemoglobin with less heme iron consumed; as the amount of dietary
iron from the HIP increased, the proportional gain in hemoglobin was less. This result is

Nutrients 2024,16, 4029 7 of 10
similar to the findings of previous studies that investigated the total percentage of dietary
iron absorption [32,36].
A study investigating the bioavailability of iron from fresh, cooked, or nitrosylated
hemoglobin to anemic rats found similar, albeit slightly lower, HRE values for their
hemoglobin product [
34
], especially when compared to the HRE values we obtained at the
12 mg iron/kg diet level. This study also found that the efficiency of hemoglobin regener-
ation in anemic rats fed nitrosylated hemoglobin was lower compared to unnitrosylated
products and that cooking, as in our porcine sample, did not affect the availability of the
heme iron [
34
]. The influence of other dietary inhibitors and enhancers of iron absorption,
especially affecting non-heme iron absorption, has been well studied, including exten-
sive reviews [
37
,
38
]. In countries with high meat consumption, heme iron may comprise
one-third of total dietary iron yet account for two-thirds of the iron absorbed by the body,
attributed to the selective preference for heme iron absorption, since it remains soluble
in the small intestine, and because heme iron absorption by enterocytes is not adversely
affected by dietary inhibitors [
37
,
38
]. Therefore, the use of HIP as a food fortificant may
favorably overcome the dietary inhibition caused by phytate, tannins, and calcium, which
is often observed when testing the bioavailability of inorganic iron.
One limitation of this study may be that additional concentrations of dietary iron from
the HIP (that is, <12 or >48 mg iron/kg diets) were not used. However, our study design,
which included four distinct graded (increasing) concentrations of dietary iron, reflects a
design approach that has been used to test a variety of other forms of iron fortificants. Hence,
the four iron concentrations used in this study were used as a framework for comparing
our results to other studies in the field of iron absorption and assessment of HRE using
murine models. Another limitation may be that we used rats, whereas some studies used
mice or species other than rodents. However, because most other animal models of human
iron absorption have been murine models, especially rats, this study was designed to
enable further comparisons to previous research using the hemoglobin repletion assay; it is
important to note that this assay typically uses a threshold for selecting rats for repletion
treatment set at a lower level of anemia, considering that in humans a value of 6 g/dl Hb
is the threshold for blood transfusion and anemia is usually assumed at values <10 g/dL.
Further, the measurement of HRE as part of this assay may not take into account the effects
of iron overload, especially at higher levels of iron in the diet. Nevertheless, other studies
have found that the efficiency of converting dietary iron into hemoglobin by anemic rats
was very similar to reported absorption values for heme iron by iron-deficient human
subjects [
34
,
37
–
39
]. Therefore, data from this study may be useful when considering new
food fortification policies and guidelines and the potential utilization of heme iron powders
as part of global micronutrient fortification programs under consideration [
22
]. Although
serum transferrin, ferritin, and total iron binding capacity (TIBC) were not measured
because the focus of this study was HRE, understanding how these indices change in the
context of hemoglobin repletion would provide beneficial insights. Additionally, because
of religious or vegetarian dietary practices, this HIP may not be suitable or accepted as a
food fortificant.
Overall, data from this study are valuable in demonstrating that the absorption of iron
from this particular HIP increases hemoglobin and thereby reduces IDA. Anemia is gener-
ally considered to be a symptom rather than a disease because a decrease in the number of
red blood cells and erythrocytic hemoglobin (mean cell hemoglobin content) can be caused
by nutritional deficiencies or a variety of underlying medical conditions, such as bleeding
disorders and chronic diseases, with thalassemia or chronic kidney diseases, respectively,
common non-nutritional etiologies. Therefore, identifying the etiology of anemia is vital for
treatment. Because current food fortification guidelines for iron, especially using non-heme
elemental iron or iron salts and fortification of staple foods (grain flours), suggest varying
the amount of iron used based on the type or form of iron, our findings are also beneficial
in illustrating the usefulness of HIP as an alternative food fortification approach that may
be effective in reducing IDA. Using the HIP as a dietary supplement may also be beneficial

Nutrients 2024,16, 4029 8 of 10
in preventing and/or minimizing IDA, reducing the need for clinical care, and thereby
helping to reduce medical costs. Although dietary therapy for anemia is a well-established
approach and iron supplements may be purchased inexpensively, the use of heme iron
powder offers an effective alternative approach and another efficacious option to acquire
and consume a form of relatively well-absorbed dietary iron and thereby help prevent IDA.
Globally, iron deficiency anemia remains one of the most common micronutrient
deficiencies [
40
–
42
]. A recommendation to help combat persistent IDA in communities
worldwide is for additional studies of HIP to include human clinical trials to determine
optimal levels of HIP in the diet, providing hematological support to individuals at risk of
developing IDA.
5. Conclusions
In summary, our findings are valuable for illustrating that this heme iron powder is
a useful fortification agent to replenish hemoglobin and resolve iron deficiency anemia.
Considering the favorable absorption profile of heme in comparison to non-heme iron,
the inclusion of heme iron powder as an iron fortifcant in a variety of staple foods may
be both efficacious and advantageous in reducing the incidence of iron deficiency and its
associated anemia.
Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/nu16234029/s1, Figure S1: Iron (Fe) intake (mg/day) of anemic
rats fed graded quantities of heme iron powder for a 14-day repletion period. Values are mean
values ±SEM
(n= 9–12/group). Different letters are used to denote significant differences (p
≤
0.05)
from higher to lower Fe intakes.
Author Contributions: Conceptualization, J.H.S.; methodology, J.H.S.; software, J.H.S.; validation,
J.H.S.; formal analysis, J.H.S.; investigation, J.H.S. and L.D.G.; resources, J.H.S.; data curation,
J.H.S. and L.D.G.; writing—original draft preparation, J.H.S. and L.D.G.; writing—review and
editing, J.H.S. and L.D.G.; visualization, J.H.S. and L.D.G.; supervision, J.H.S.; project administration,
J.H.S.; and funding acquisition, J.H.S. All authors have read and agreed to the published version of
the manuscript.
Funding: This work was supported in part, including provision of the heme iron powder, by Proliant,
Inc. (PL; Ankeny, IA, USA)/APC Europe, S.A. (APC; Barcelona, Spain) and Case Western Reserve
University (Cleveland, OH, USA); grant reference numbers: CON110654V3 (PL/APC) and HSAF-G-
RB13G (CWRU).
Institutional Review Board Statement: This study was conducted in accordance with the Institutional
Animal Care and Use Committee (IACUC) of Case Western Reserve University, Cleveland, OH, USA
(Permit number: 2011-0046; date of approval 11 January 2011).
Informed Consent Statement: Not applicable.
Data Availability Statement: The original contributions and data on which findings are presented in
this study are included within the article, as well as the supporting material that accompanies this
submission.
Acknowledgments: We gratefully acknowledge the contributions of the Case Western Reserve
University (CWRU) Animal Resource Center and Jayne Poyer and staff for their help with animal care.
Conflicts of Interest: J.H.S. received research funding from Proliant, Inc. (Ankeny, IA, USA)/APC
Europe, S.A. (Barcelona, Spain). The funders were not involved in the study design, collection,
analysis, interpretation of data, the writing of this article, or the decision to submit it for publication.
The remaining authors declare that the research was conducted in the absence of any commercial or
financial relationships that could be construed as potential conflicts of interest.
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