**Beneficial E**ff**ect of Ubiquinol on Hematological and Inflammatory Signaling during Exercise**

**Javier Diaz-Castro 1,2,\*, Jorge Moreno-Fernandez 1,2, Ignacio Chirosa 3, Luis Javier Chirosa 3, Rafael Guisado <sup>4</sup> and Julio J. Ochoa 1,2**


Received: 13 January 2020; Accepted: 4 February 2020; Published: 6 February 2020

**Abstract:** Strenuous exercise (any activity that expends six metabolic equivalents per minute or more causing sensations of fatigue and exhaustion to occur, inducing deleterious effects, affecting negatively different cells), induces muscle damage and hematological changes associated with high production of pro-inflammatory mediators related to muscle damage and sports anemia. The objective of this study was to determine whether short-term oral ubiquinol supplementation can prevent accumulation of inflammatory mediators and hematological impairment associated to strenuous exercise. For this purpose, 100 healthy and well-trained firemen were classified in two groups: Ubiquinol (experimental group), and placebo group (control). The protocol was two identical strenuous exercise tests with rest period between tests of 24 h. Blood samples were collected before supplementation (basal value) (T1), after supplementation (T2), after first physical exercise test (T3), after 24 h of rest (T4), and after second physical exercise test (T5). Hematological parameters, pro- and anti-inflammatory cytokines and growth factors were measured. Red blood cells (RBC), hematocrit, hemoglobin, VEGF, NO, EGF, IL-1ra, and IL-10 increased in the ubiquinol group while IL-1, IL-8, and MCP-1 decreased. Ubiquinol supplementation during high intensity exercise could modulate inflammatory signaling, expression of pro-inflammatory, and increasing some anti-inflammatory cytokines. During exercise, RBC, hemoglobin, hematocrit, VEGF, and EGF increased in ubiquinol group, revealing a possible pro-angiogenic effect, improving oxygen supply and exerting a possible protective effect on other physiological alterations.

**Keywords:** high intensity exercise; ubiquinol; hematological parameters; inflammation; ergogenic effect

#### **1. Introduction**

There are multiple beneficial effects associated with regular and planned exercise [1,2], including the reduction in the age-related changes in nuclear pore complex proteins, protection of the neuromuscular junction, and the increase in the lives of susceptible motoneurons, preserving neuromuscular integrity and innervation status [3,4]. In addition, exercise increases blood flow and improves vascular integrity, enhancing angiogenesis, resulting in reversal of rarefaction and hypertension, and enhancement of cerebral blood flow and cognition [5].

On the other hand, strenuous exercise can induce a range of adverse effects including oxidative stress, hematological changes, and inflammatory response involved in activating catabolic pathways inducing muscle damage [6]. Strenuous exercise (defined as any activity that expends six metabolic equivalents (METS) per minute or more [7]) is harmful to health [8], because it causes structural damage to muscle cells indicated by muscle soreness and swelling, prolonged loss of muscle function, increased free radicals output, induction of the pro-inflammatory signaling, impairment of immune functions, including the immunoglobulin production or T-cell function, and leakage of muscle proteins into circulation, among other effects [9,10].

The aerobic energy metabolism during strenuous exercise (established when sensations of fatigue and exhaustion occur, inducing deleterious effects, affecting negatively different cells) plays a crucial role on the performance. Hematological changes such as decreases in the hemoglobin (Hb) concentrations and RBC counts are often found to result from participation in strenuous exercise [11]. In this sense, in athletes performing high intensity exercise, a high prevalence of "sports anemia" or iron deficiency anemia induced by strenuous exercise has been reported [12,13], which has been associated with hematological changes such as a decrease in RBC, hemoglobin, and hematocrit [14,15]. These changes are suggested to be mainly caused by iron deficiency and a negative iron balance caused by intense physical exercise [16].

Within the oxygen transport chain, RBC mass is crucial for oxygen supply to working muscles which could regulate the aerobic performance capacity [17]. However, although during the strenuous exercise there is a greater erythropoietic activity, an important loss of RBC is featured [14,18]. During exercise an inflammatory state is featured and a high production of free radicals, both factors affecting iron metabolism [18] and by a direct effect on the iron reduction due to its affinity for H2O2 [13]. In addition, skeletal muscle is a highly regenerative tissue, but muscle repair potential is limited, and inflammatory signaling contributes to muscle repair [19], however the pro-inflammatory cytokines can also be deleterious to health. Therefore, these cytokines are both cause and effect of inflammation [20]. Elevated levels of these inflammatory markers not only increase risk for chronic diseases, but also contribute to disease pathogenesis [21]. In addition, the composition of the serum microbiome is linked to indices of inflammation, altering immunity [22].

Athletes may be susceptible to a heightened anti-inflammatory state. These results can transiently suppress immune function and increase the risk of infection [23]. In this sense, reducing inflammation has been recognized as one of the ways to reduce the risk of chronic disease [24].

During regeneration, reconstitution of muscle fibers, RBC mass, and blood supply is imperative for full muscular recovery and prevention of muscle atrophy [25]. In this sense, vascular endothelial growth factor (VEGF) has a key role for angiogenesis and muscle fibers repair in skeletal muscle and has been shown to be upregulated by a single bout of dynamic exercise [26].

On the other hand, inflammation, angiogenesis, RBC mass, and oxygen supply have a key role on muscle damage associated to high intensity exercise and other physiological alterations that can affect physical performance. Therefore, it would be interesting to assess the effect of oral supplementation with a substance capable of improving hematological parameters and diminishing inflammatory signaling associated to this performance [27,28], however scarce studies are available about molecules with these characteristics to support the regeneration process of skeletal muscle after strenuous exercise. One of these substances could be coenzyme Q10 (CoQ10) [29].

The data available in the scientific literature have provided a direct link between physical performance and blood and muscle tissue CoQ10 levels [30]. However, most of these studies are focused mainly on the exercise performance and radical-scavenging activity of CoQ10 during low intensity exercise [30], with the studies about the influence of CoQ10 supplementation during the performance of high intensity (strenuous) exercise on the inflammatory signaling, hematology, and muscle recovery after strenuous exercise being scarce. CoQ10 exists in two forms: Ubiquinone (oxidized form), the most common form in CoQ10 supplements, and ubiquinol, the reduced and most active form, which has properties related to bioenergetic and antioxidant activity [31,32], but poorly studied. Therefore, we aimed to determine whether, a short term oral ubiquinol supplementation may be efficient ameliorating the pro-inflammatory effects, improving hematological parameters, effects that could promote skeletal muscles regeneration and oxygen supply after strenuous exercise.

#### **2. Materials and Methods**

#### *2.1. Subjects and Supplementation Protocol*

This study was a randomized, double-blind, and placebo-controlled trial. One hundred healthy and well trained, but not on an elite level, firemen of the Fire Department of the City of Granada were taking part in this study. Participants completed a medical and health history and physical activity questionnaire (IPAQ-SF) [33] prior to enrolment. All of them were nonsmokers, did not take any nutritional supplements and did not present febrile/inflammatory clinical symptoms, did not use immunosuppressive or nephrotoxic drugs, did not use energy, protein, and/or antioxidant supplements. The firemen were randomly divided into two groups: Ubiquinol group (ubiquinol) (n = 50), and placebo group (control) (n = 50). The ubiquinol group was supplemented with an oral dose of 200 mg/day of ubiquinol during two weeks, administrating two brown liquid filled hard gelatine capsules of 100 mg/day, and subjects assigned to the control group took placebo using the same scheme. The capsules Kaneka QH ubiquinol (Kaneka Corporation, Osaka, Japan) contained 100 mg of ubiquinol in a basis of canola oil, diglycerol monooleate, beeswax, and soy lecithin. The placebo capsules contained the same composition without ubiquinol and were also supplied by Kaneka (Kaneka Corporation, Osaka, Japan). The study was approved by the Commission of Ethics in Human Research of the University of Granada (ref. 804). The study has been registered in ClinicalTrials.gov, with number NCT01940627. Informed consent was obtained from all subjects with written consent to participate in this study.

#### *2.2. Strenuous Exercise Performance Programme*

Characteristics, intensity, and muscle damage (loss of skeletal muscle function and soreness) of this protocol was previously reported by measuring blood myoglobin and CK [32] and similar increases have been observed in other strenuous exercise tests [34]. After two-weeks period of ubiquinol or placebo supplementation, subjects performed the strenuous exercise protocol in order to induce muscle damage. Prior to the starting of each test, subjects performed a warm-up which was divided into two phases: General activation phase and specific phase. The protocol consisted of conducting two identical strenuous exercise tests, with a rest period between tests within 24 h. Circuit weight training (CWT), characterized by alternating exercises between upper- and lower-body segments performed at stations, has been widely used in practice settings. The main advantage of this method is that it allows faster training performance. Moreover, it is commonly used for persons interested in weight management, although a previous study showed similar effects on body composition and on muscular strength and size in trained men after CWT and multiple-set resistance training [35]. The rest time between sets was five minutes to allow recovery and complete the designed workout [35]. Both strenuous exercise tests consisted of performing a circuit composed of 10 bodybuilding exercises (1. athletic press; 2. chest press in Smith Machine; 3. seated oar; 4. shoulders press; 5. femoral biceps flexion; 6. chest press in Smith Machine; 7. step with weight; 8. surveyor's pole chest; 9. shove with weight; 10. quadriceps extension) [32]. In order to establish the minimum magnitude of the load to be displaced for each subject, one week before strenuous exercise protocol, a session of pre-training was held with the subjects to conform the load individually in terms of two parameters in each exercise: (a) Scale OMNI-RES [36] values of perceived exertion between 6–7, and (b) 10 repetitions.

#### *2.3. Blood Sampling*

Blood samples were collected from the participants by venous catheter into heparinized tubes before and immediately after the physical test. Five blood samples and urine were taken: Before supplementation (basal value) (T1), after supplementation (two weeks) (T2), after first physical exercise test (T3), after 24 h of rest (T4), after second physical exercise test (T5). One aliquot of blood was collected in tubes with an EDTA anticoagulant for hematological analysis. The remaining blood was immediately centrifuged at 1750 g for 10 min at 4 ◦C in a Beckman GS-6R refrigerated centrifuge (Beckman, Fullerton, CA, USA) to separate plasma from red blood cell pellets. Plasma samples were immediately frozen and stored at −80 ◦C until analysis.

#### *2.4. Inflammatory Parameters*

Epidermal growth factor (EGF), interferon gamma (IFN-γ), vascular endothelial growth factor (VEGF), monocyte chemotactic protein 1 (MCP-1), tumor necrosis factor alpha (TNF-α), interleukin (IL)-1, IL-1ra (receptor agonist), IL-6, IL-10, and IL-15 were determined using the HCYTOMAG-60K Milliplex MAP Human Cytokine/Chemokine Magnetic Bead Panel (Millipore Corporation, Missouri, USA), based on immunoassays on the surface of fluorescent-coded beads (microspheres), following the specifications of the manufacturer (50 events per bead, 50 μL sample, gate settings: 8000–15,000, time out 60 s, melatonin bead set: 34). The plate was read on a LABScan 100 analyzer (Luminex Corporation, Austin, TX, USA) with xPONENT software for data acquisition. With these biomarkers we characterize mediators of adaptive immunity, mediators of innate immunity and inflammation, chemotaxis, haematopoietic mediators and growth factors, allowing us to have an overview of the various pathways of cytokines in the immune and inflammatory process. Average values for each set of duplicate samples or standards were within 15% of the mean. Cytokines concentrations in plasma samples were determined by comparing the mean of triplicate samples with the standard curve for each assay.

#### *2.5. Hematological Parameters*

Hemoglobin (Hb) concentration, red blood cells (RBC), hematocrit, mean corpuscular volume (MCV), mean corpuscular Hb (MCH), mean corpuscular Hb concentration (MCHC), red cell distribution width (RDW), platelets, mean platelets volume (MPV), leukocytes, neutrophils, lymphocytes, monocytes, eosinophils, basophils of fresh blood samples were measured using an automated hematology analyzer Mythic 22CT (C2 Diagnostics, Grabels, France).

#### *2.6. Statistical Analysis*

All data are presented as the mean ± standard error of the mean (SEM). All variables were tested to see if they followed the criteria of normality and homogeneity of variance using the Kolmogorov–Smirnoff's and Levene's tests, respectively. To compare general characteristics of the subjects in both experimental groups, unpaired Student's t-test was used. To assess the effect of the supplementation and the evolution in the time of each variable studied in each experimental group a general linear model of variance for repeated measures with an adjustment by means of Bonferroni´s test has been performed. Bonferroni's test allowed us to know intra- and inter-subject differences (effect of time in each group and supplementation in each period, respectively) in a very robust way in terms of power. A value of *p* < 0.05 was considered significant. For data analysis we used the SPSS version 20.0 (SPSS Statistics for Windows, 20.0.0. SPSS INC. Chicago, IL, USA).

#### **3. Results**

No statistically significant differences between both groups were found for weight, age, height, and BMI (Table 1). In addition, no significant differences were recorded between groups for the short form of the International Physical Activity Questionnaire [32]. The subjects of both experimental groups were categorized as "Health Enhancing Physical Activity" (HEPA Active): Category 3, the highest measurement threshold of total physical activity of the questionnaire. In addition, as we have previously reported [32], the high intensity protocol induces muscle damage based on lactate output (increase of 290% (2.9 ± 0.1 vs. 8.5 ± 0.3 mmol/L) after the first exercise session and an increase of 355% (2.9 ± 0.1 vs. 10.5 ± 0.5 mmol/L) after the second training session, myoglobin increased 358% higher after the first session (25.7 ± 3.2 vs. 92.1 ± 7.9 ng/mL) and 387% after the second session (25.7 ± 3.2 vs. 99.3 ± 6.8 ng/mL) and creatine kinase (CK-MM) increased 158% after the first exercise test (2.01 ± 0.3 vs. 3.2 ± 0.4 ng/mL) and 196% after the second session (2.01 ± 0.3 vs. 4.0 ± 0.3 ng/mL). We have also reported that

ubiquinol supplementation increased plasma CoQ10 levels 522% (1.00 ± 0.06 vs. 5.22 ± 0.41 mmol/L). The dropout percentage was similar in both groups (24% after finishing the first test and 32% after finishing the second exercise test) and neither differences were observed between dropout reasons in both groups [32].


**Table 1.** Subjects baseline characteristics.

Data expressed as the mean ± SEM. BMI: Body mass index; SBP: Systolic blood pressure; DBP: Diastolic blood pressure; RHR: Resting heart rate.

IL-1 was lower in ubiquinol compared with the control group in T3. Regarding the evolution, IL-1 increased in the ubiquinol and control group in T5 compared with T1, T2, T3, and T4 (Figure 1A). No differences were observed in IL-1ra due to the supplementation. Regarding the evolution, IL-1ra increased in the ubiquinol group in T5 compared to T4, T2, and T1 (Figure 1B). No differences were observed in IL-6 due to the supplementation. Regarding the evolution, IL-6 increased in the ubiquinol group in T3 and T5 compared with T1, in T3 and T5 compared with T2, and also in T5 compared with T4, while decreased in T4 compared with T3 (Figure 1C). IL-8 was lower in the supplemented group in T3 compared with the control group. Regarding the evolution, in the ubiquinol group, IL-8 decreased in T4 with respect to T2, T3, and T5. In the control group, there was an increase in T3 with respect to T1, T2, and T4, and also increased in T5 with respect to T1 and T4 (Figure 1D). IL-10 was higher in ubiquinol compared with the control group in T5 (Figure 1E). Regarding the evolution, IL-10 increased in the ubiquinol group in T3 compared with T1 and T5, and also increased in T5 with respect to T1 and T4 while no differences by the evolution of time were observed in the control group. No differences were observed in IL-15 due to the supplementation. Regarding the evolution, IL-15 decreased in T4 compared with T3 in the control group (Figure 1F).

**Figure 1.** Effects of exercise and ubiquinol supplementation on plasma cytokines: interleukin (IL)-1 (**A**), IL-1ra (**B**), IL-6 (**C**), IL-8 (**D**), IL-10 (**E**), IL-15 (**F**). Results are expressed as the mean ± SEM. \* means statistically significant differences between groups (*p* < 0.05). T1: Before supplementation (basal value); T2: After supplementation (two weeks) and before the first physical test; T3: After first physical exercise test; T4: After 24 h of rest and before the second physical test; T5: After second physical exercise test. Different letters in every group indicates significant differences due to the time (control (A, B, C, D, E); ubiquinol (a, b, c, d, e)) (*p* < 0.05).

No differences were observed in TNF-α due to the supplementation. Regarding the evolution, TNF-α increased in the ubiquinol group in T3 compared with T1 and T4. In the control group, TNF-α increased in T3 compared with the rest of the blood samples and also increased in T5 compared with T1 and T4 (Figure 2A). No differences were observed in IFN-γ due to the supplementation or evolution of time (Figure 2B). A higher level of EGF was observed in the ubiquinol compared with the control group in T2 and T3. Regarding the evolution, EGF increased in the ubiquinol group in T2 compared with T1 and in T3 compared with T1, T4, and T5. In the control group, EGF was higher in T1 with regard to T1, T2 and also increased in T4 with regard to T1 (Figure 2C). VEGF was higher in the ubiquinol compared with the control group in T3. VEGF increased in the ubiquinol group in T5 compared with T1 and T2, while no differences by the evolution of time were observed in the control group (Figure 2D). MCP-1 decreased in ubiquinol compared with the control group in T3 and T5. Regarding the evolution, MCP-1 increased in the ubiquinol group in T3 compared with T1, and in T5 compared with T4, while decreased in T4 compared with T2, and in T4 compared with T3. In the control group, MCP-1 increased in T2, T3, and T5 compared with T1, increased in T3 compared with T2, and in T5 compared with T4, while showed a decrease in T4 compared with T2, and in T4 and in T5 compared with T3 (Figure 2E).

**Figure 2.** Effects of exercise and ubiquinol supplementation on plasma cytokines: tumor necrosis factor alpha (TNF-α) (**A**), interferon gamma (IFN-γ) (**B**), epidermal growth factor (EGF) (**C**), vascular endothelial growth factor (VEGF) (**D**), monocyte chemotactic protein 1 (MCP-1) (**E**). Results are expressed as mean ± SEM. \* means statistically significant differences between groups (*p* < 0.05). T1: Before supplementation (basal value); T2: After supplementation (two weeks) and before the first physical test; T3: After first physical exercise test; T4: After 24 h of rest and before the second physical test; T5: After second physical exercise test. Different letters in every group indicates significant differences due to the time (control (A, B, C, D, E); ubiquinol (a, b, c, d, e)) (*p* < 0.05).

RBC increased in the ubiquinol compared with the control group in T3 and T4. Regarding the evolution, RBC increased in the ubiquinol group in T2 and T3 compared with T1 and T5, while decreased in the control group in T4 and T5 compared with T1 (Figure 3A). Hemoglobin levels were higher in ubiquinol compared with the control group in T3 and T4. In the control group, hemoglobin decreased in T4 compared with T1, T2, T3, and T5 (Figure 3B). Hematocrit increased in the ubiquinol compared with the control group in T3. Hematocrit decreased in T4 and T5 compared with T1 in the control group (Figure 3C).

Leukocytes increased in the ubiquinol group in T3 compared with T1 and T4. Neutrophils were higher in the ubiquinol compared with the control group in T4, also increased in the control group in T5 compared with T1, T2, and T4 and also in T3 and T5 in regard to T1, T2, and T4 in the ubiquinol group. Lymphocytes decreased in the ubiquinol group in T4, in T5 compared with T1 and T2 in the ubiquinol group and in T3, and also in T5 compared with T4 in the control group. Monocytes were lower in the ubiquinol compared with the control group in T4, also decreased in T2, T3, T4, and T5 compared with T1 in the control and ubiquinol group. Eosinophils decreased in T3 compared with T1 in the control and ubiquinol group. Basophils decreased in T4 and T5 compared with T1 in the ubiquinol group and increased in T5 compared with T1 and T4 in the control group. No changes were recorded in platelets during the study, MCV or MCH nor due to exercise and neither to ubiquinol. MCHC increased in the ubiquinol compared with the control group in T5 and also increased in T4 and T5 compared with T1 and T2 in the ubiquinol group, while decreased in T3, T4, and T5 compared with T1 in the control group. No significant changes were recorded in RDW and MPW due to exercise and neither to ubiquinol (Table 2).

**Figure 3.** Effects of exercise and ubiquinol supplementation on hematological parameters: Red blood cells (**A**), hemoglobin (**B**), hematocrit (**C**). Results are expressed as mean ± SEM. \* means statistically significant differences between groups (*p* < 0.05). T1: Before supplementation (basal value); T2: After supplementation (two weeks) and before the first physical test; T3: After first physical exercise test; T4: After 24 h of rest and before the second physical test; T5: After second physical exercise test. Different letters in every group indicates significant differences due to the time (control (A, B, C, D, E); ubiquinol (a, b, c, d, e)) (*p* < 0.05).

**Table 2.** Effects of exercise and ubiquinol supplementation on hematological parameters. Results are expressed as mean ± SEM. \* means statistically significant differences between groups (*p* < 0.05). T1: Before supplementation (basal value); T2: After supplementation (two weeks) and before the first physical test; T3: After first physical exercise test; T4: After 24 h of rest and before the second physical test; T5: After second physical exercise test. Different letters in every group indicates significant differences due to the time (control (A, B, C, D, E); ubiquinol (a, b, c, d, e)) (*p* < 0.05).


MCV: Mean corpuscular volume; MCH: Mean corpuscular hemoglobin; MCHC: Mean corpuscular hemoglobin concentration; RDW: Red cell distribution width; MPV: Mean platelet volume.

#### **4. Discussion**

Regular physical exercise is associated with numerous health benefits including a lower risk of all-cause mortality [37,38], nevertheless, strenuous exercise specially in amateur athletes, promotes the generation of oxidative stress and a pro-inflammatory state, which are one of the main reasons for the muscular aggression observed in high intensity exercise, together with other physiological alterations such as sports anemia [12], characterized by hematological changes including decrease in RBC, hemoglobin, and hematocrit [12,13,15]. These changes reduce oxygen supply and energy production which could regulate the aerobic performance capacity [17], which could lead to a reduction in the physical performance, incorrect adaptation to training protocols, and other possible physiological alterations [12]. Taking into account the importance of inflammation in most of these alterations, supplementation with effective molecules against these alterations could be beneficial. CoQ10 could be suitable for a muscle-protective supplementation because it has anti-inflammatory and antioxidant activity and it is intimately involved in energy production [39,40]. However, scarce studies of CoQ10 supplementation investigating its effects during strenuous physical exercise are available in the scientific literature, especially in the field of the inflammatory signaling and hematological parameters and virtually nonexistent when referring to supplementation with the reduced form of this molecule (ubiquinol) [29,39]. Both groups studied were homogeneous in terms of weight, height, blood pressure, and age. In addition, as commented above, we have previously reported that both groups showed similar physical nutritional status and characteristics, and that our protocol of exercise features a high intensity and induced muscle damage [32].

During intense exercise, muscles are damaged and reconstitution of muscle fibers is imperative for full muscular recovery and prevention of muscle atrophy, being pivotal factors in this process the inflammatory signaling and an adequate RBC mass and blood supply. The inflammatory response during high intensity exercise initiates a rapid and sequential invasion of muscle fibers, mediating the repairing process during recovery from strenuous exercise. Thus, the inflammatory response induced by muscle damage may be a functionally response to favor muscle regeneration [41].

In response to exercise, skeletal muscle releases pro-inflammatory cytokines exponentially according to the exercise intensity, duration, mass of muscle recruited, and endurance capacity [42]. Acute bouts of exercise cause transient damage to contracting skeletal muscles, triggering an inflammatory response that increases the levels of pro-inflammatory cytokines and acute-phase reactants in the blood [43], such as IL-6 and TNF-α which are pro-inflammatory cytokines primarily secreted by stimulated immune cells (e.g., monocytes and macrophages) [44], nevertheless, although IL-6 and TNF-α are pro-inflammatory agents, they can stimulate the production of an anti-inflammatory cytokine such as IL-10 [45]. In the ubiquinol group, the increase in IL-6 is not preceded by an increase in tumor necrosis factor alpha (TNF-α) and, most importantly, is followed by increased levels of anti-inflammatory cytokines, namely IL-1 receptor antagonist (IL-1ra) and IL-10 [45,46], together with a decrease in IL-8, as well as lower expression of Il-1, IL-15, and MCP-1. In turn, IL-10 inhibits the synthesis of some pro-inflammatory cytokines such as TNF-α [47], a fact that can be observed especially after the second physical test. TNF-α seems to have a biphasic effect on muscle: High levels of the cytokine promote muscle catabolism, probably by a nuclear factor kB (NF-kB) mediated effect, whereas low levels of TNF-α such as those recorded in the ubiquinol group do not induce NF-kB and stimulate myogenesis [48].

During strenuous exercise, there is a reduction in RBC, which could imply a decrease in the oxygen supply to the cells. On the other hand, previous investigations have reported that lower cardiorespiratory fitness, assessed by maximal oxygen consumption (VO2max kg<sup>−</sup>1), is associated with higher basal IL-8 and MCP-1 concentrations [49,50]. High VO2max kg−<sup>1</sup> is correlated with low IL-8 levels [51], suggesting that low IL-8 levels is an important inflammatory parameter that predicts the level of cardiorespiratory fitness. In the current study, ubiquinol also reduced MCP-1 (key chemokine that regulates migration and infiltration of monocytes/macrophages in response to inflammation) and IL-8. Interestingly, at physiological concentration, MCP-1 is the only adipocytokine able to impair insulin signaling and glucose uptake in skeletal muscle and in this sense, a decrease in this biomarker during physical exercise facilitates insulin-mediated glucose disposal [52]. Therefore, we can assume that rather than pro-inflammatory, the acute exercise-induced increase in pro-inflammatory cytokines such as IL-6, in the ubiquinol group, may actually lead to an anti-inflammatory environment [53] and also an improvement in physical performance due to a higher cardiorespiratory fitness and glucose consumption.

Regarding the hematological changes associated with strenuous exercise, it is asserted that exercise performed until exhaustion decreases the number of leukocytes, a fact that can be associated with metabolic changes such as ischemia that occur during exercise and increased muscle activity leads to a greater incidence of capillary swelling and leukocyte adherence to venules [54]. However, as explained below, ubiquinol features a vasodilator activity and pro-angiogenic effect which avoids capillary swelling and leukocyte adherence to blood vessels, reducing impairment of immune functions related to strenuous exercise [55], avoiding this reduction in the leukocytes. Moreover, as mentioned above, ubiquinol reduced MCP-1 that plays an important role in selectively recruiting monocytes and lymphocytes [56], the reason why we recorded a decrease of these white cells after 24 h of the first physical test.

As previously mentioned, in elite athletes, a decrease in the percentages of RBC, hemoglobin and hematocrit is recorded and, in this situation, inflammation is one of the main causes. Even if there is only a small decrease in RBC, it is important to highlight that during strenuous exercise a greater demand for oxygen is necessary and therefore even a small decrease in RBC could affect the performance. Thus, an adequate RBC mass and blood supply is imperative for full muscular recovery, energy requirements, and prevention of muscle atrophy [25]. In this regard, there are three aspects that have to be taken into account: The vasodilation, the angiogenesis or generation of new blood vessels, and the RBC mass circulating, physiological pathways that are closely related to increase blood supply. In the current study, ubiquinol prevented the decrease in RBC, hemoglobin, and hematocrit after the first physical test, a fact that would increase oxygen delivering to tissues, especially muscles. During exercise, oxidative stress induces deleterious structural and functional changes in RBC, however, ubiquinol prevents those changes in erythrocytes, due to its antioxidant properties [29], avoiding the impairment in hematological changes such as decreases in Hb and RBC caused by strenuous exercise [11]. In addition, ubiquinol supplementation also increases NO output [32], a fact that is beneficial during physical activity, featuring a vasodilator action that helps both exercise performance and nutrient supply in muscle recovery, as well as improvement in the supply of substrates such as glucose, together facilitate the regulatory role in the immune system [57]. This can be explained by the link between VEGF and nitric oxide (NO). VEGF is a critical cytokine involved in angiogenesis, and NO is a downstream effector. Importantly, recent studies suggest that NO is an essential mediator of endothelial cells migration and VEGF-induced angiogenesis [58]. Angiogenesis is a crucial process for effective regeneration not only by providing stable vessels for supporting the metabolic activity of the regenerated tissue, but also by generating a new population of endothelial and periendothelial cells that will supply a large array of molecules that sustain myogenesis [25]. In this sense, EGF promotes growth and migration of vascular smooth muscle cells through activation of EGF receptor, and VEGF promotes the generation of new blood vessels, essential processes during vascular remodeling and muscular fibers reconstitution [59,60]. In our study, VEGF increased in the ubiquinol group, revealing not only a vasodilator action of ubiquinol due to the NO, but also a pro-angiogenic effect, a fact that would improve nutrient and oxygen supply and muscle recovery after strenuous exercise, supporting and explaining the possible ergogenic effect of ubiquinol during intense exercise. On the other hand, ubiquinol also increased EGF, probably due to its antioxidant activity [61]. VEGF and EGF increase due to the ubiquinol supplementation can be directly linked with the muscle fibers regeneration after intense exercise.

#### **5. Conclusions**

In summary, the present study demonstrates a strong correlation between the high intensity exercise and inflammatory signaling as shown by the overexpression in the pro-inflammatory cytokines. In addition, the present findings provide evidence that oral supplementation of ubiquinol during high intensity exercise could modulate the inflammatory signaling associated to exercise by reducing the overexpression of pro-inflammatory cytokines, together with an increase in anti-inflammatory cytokines which limits the detrimental, pro-inflammatory actions of strenuous exercise. In addition, during exercise, RBC, hemoglobin, hematocrit, VEG, NO, and EGF did not decrease in the ubiquinol group, revealing a possible pro-angiogenic effect, a fact that could improve nutrients and oxygen supply and therefore muscle recovery after strenuous exercise. Therefore, the knowledge gained from these findings reveals the benefit of ubiquinol supplement in athletes prior to the performance of strenuous exercise in order to reduce the undesirable effects of the inflammation signaling during high intensity exercise, increasing blood supply, reducing the muscle damage and hematological impairment, and improving skeletal muscle fibers regeneration.

**Author Contributions:** J.J.O. designed the research proposal and provided funding. J.D.-C. and J.M.-F. conducted the research and wrote the manuscript. I.C., L.J.C., and R.G. revised the data and the manuscript. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. All authors have read and agree to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** J.M.-F. was supported by a fellowship from the Ministry of Education, Culture and Sport (Spain), and is grateful to the Excellence PhD Program "Nutrición y Ciencias de los Alimentos" from the University of Granada. We would like to thank the Fire Department of Granada for their participation in the current study. Funding for this work, ubiquinol, and placebo capsules were provided by (Kaneka Corporation, Osaka, Japan). The authors are grateful to Susan Stevenson for her efficient support in the revision of the English language.

**Conflicts of Interest:** The investigators and the University of Granada have no direct or indirect interest in the tested product (Kaneka QH) or in Kaneka Corporation and therefore the authors declare no conflict of interest.

#### **References**


© 2020 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 (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Iron Status of Infants in the First Year of Life in Northern Taiwan**

#### **Chiao-Ming Chen 1,**†**, Shu-Ci Mu 2,3,**†**, Chun-Kuang Shih 4, Yi-Ling Chen 3,5, Li-Yi Tsai 3,6, Yung-Ting Kuo 7,8, In-Mei Cheong 1, Mei-Ling Chang 1, Yi-Chun Chen <sup>4</sup> and Sing-Chung Li 4,\***


Received: 3 December 2019; Accepted: 31 December 2019; Published: 3 January 2020

**Abstract:** Iron deficiency (ID) and iron deficiency anemia (IDA) typically occur in developing countries. Notably, ID and IDA can affect an infant's emotion, cognition, and development. Breast milk is considered the best food for infants. However, recent studies have indicated that breastfeeding for more than six months increases the risk of ID. This study investigated the prevalence of ID and IDA, as well as the association between feeding type and iron nutritional status in northern Taiwan. A cross-sectional study was conducted on infants who returned to the well-baby clinic for routine examination from October 2012 to January 2014. Overall, 509 infants aged 1–12 months completed the iron nutritional status analysis, anthropometric measurement, and dietary intake assessment, including milk and complementary foods. The results revealed that 49 (10%) and 21 (4%) infants in their first year of life had ID and IDA, respectively, based on the World Health Organization criteria. Breastfed infants had a higher prevalence rate of ID and IDA than mixed-fed and formula-fed infants (*p* < 0.001). Regarding biomarkers of iron status, plasma hemoglobin (Hb), ferritin, and transferrin saturation (%) levels were significantly lower in ID and IDA groups. The prevalence of ID and IDA were 3.7% and 2.7%, respectively, in infants under six months of age, but increased to 20.4% and 6.6%, respectively, in infants above six months of age. The healthy group had a higher total iron intake than ID and IDA groups, mainly derived from infant formula. The total dietary iron intake was positively correlated with infants' Hb levels. Compared with formula-fed infants, the logistic regression revealed that the odds ratio for ID was 2.157 (95% confidence interval [CI]: 1.369–3.399) and that for IDA was 4.196 (95% CI: 1.780–9.887) among breastfed infants (*p* < 0.001) after adjusted for all confounding factors (including gestational week, birthweight, sex, body weight percentile, body length percentile, age of infants, mothers' BMI, gestational weight gain, education level, and hemoglobin level before delivery). In conclusion, our results determined that breastfeeding was associated with an increased the prevalence of ID and/or IDA, especially in infants above six months. This suggests that mothers who prolonged breastfeed after six months could provide high-quality iron-rich foods to reduce the prevalence of ID and IDA.

**Keywords:** infant; breast milk; formula milk; iron deficiency; iron deficiency anemia

#### **1. Introduction**

Breast milk is considered the optimal nutrition and healthy food for the first six months of life, and breastfeeding is typically complemented with other foods from six months of age until at least 12 months for nearly all infants [1]. A reappraisal of the evidence from a recent expert review for the European Food Safety Authority (EFSA) concluded that for infants across the EU, complementary foods might be introduced safely between the fourth and sixth month [2]. Studies have determined that the iron content in breast milk is low, infants fed with solid food before six months or received iron supplement will decrease the risk of IDA [3]. Anemia is defined as a reduced erythrocyte count or hemoglobin (Hb) value of 5 percentile below the normal hemoglobin value specified for that age in healthy individuals [4]. Therefore, the US Department of Agriculture and the Centers for Disease Control recommend the introduction of complementary food in infants aged between 4 and 6 months [5].

In addition to inadequate iron intake, reduced bioavailability of dietary iron, increased iron requirements, and chronic blood loss is common causes of iron deficiency (ID). ID is currently the most common micronutrient deficiencies worldwide, and a World Health Organization (WHO) survey in 2008 revealed that globally, approximately 293 million (47.4% prevalence) preschool-age children and approximately 56 million (41.8% prevalence) pregnant women suffer from anemia, among which approximately 50% cases are attributable to ID [6]. ID is undoubtedly the major cause of most anemia cases, although other minerals or vitamins can also be responsible for this pathology. Prolonged ID often leads to iron deficiency anemia (IDA) [7]. The American Academy of Pediatrics determined that IDA adversely affects an infant's social and emotional behavior development and cognitive performance [8]. Approximately 25% of infants in developing countries have IDA, and IDA during pregnancy increases the risk of preterm delivery and adverse perinatal outcomes, such as maternal hemorrhage, sepsis, low birth weight, and possibly poor neonatal health [9–11].

Recent studies have indicated that iron content in breast milk is low [12–14], and prolonged use of breast milk as the main food in infants might increase the risk of ID and IDA [15]. In Taiwan, the Ministry of Health and Welfare promotes the benefits of breastfeeding for a long period. Although Tsai et al. reported that IDA was associated with prolonged, predominant breastfeeding, the study sample size was small, and no relevant risk factors were evaluated [16]. The purpose of the present study was to investigate the iron status of infants in their first year of life and analyze the relevant influencing factors.

#### **2. Participants and Methods**

#### *2.1. Study Subjects*

A cross-sectional study was conducted in three hospitals, namely, Shin Kong Wu Ho-Su Memorial Hospital, Taipei Medical University Hospital, and Shuang Ho Hospital, from October 2012 to January 2014. Overall, 2804 healthy infants aged 1–12 months who came to the well-baby clinic for routine vaccination were screened for eligibility. The inclusion criteria for infants and mothers enrolled were as follows: No systemic diseases (toxemia, hypertension, diabetes mellitus, and heart disease) during pregnancy, age below one year, healthy and without any disease, as determined by a pediatrician. Infants with premature birth, congenital diseases (such as heart, lung, liver, and intestinal diseases), growth disorders, diagnosis of gastrointestinal disorders (nausea, vomiting, pain, flatulence, diarrhea, and malabsorption), and thalassemia were excluded.

The Ethics Committee of Shin Kong Wu Ho-Su Memorial Hospital and Taipei Medical University approved this study in accordance with the International Ethical Guidelines for Biomedical Research Involving Human Subjects and ethical principles of the Declaration of Helsinki (20120901R and

201308004). Written informed consent was obtained from all participants or legal representatives before the study procedures were performed.

#### *2.2. Basic Characteristics and Dietary Iron Intake Assessment*

The contents of the questionnaire were validated by experts, including the infant's basic information, such as gestational weeks, chronological age, birth weight, birth length; anthropometrics, such as body length, body weight, and head circumference; and dietary intake. Gestational weeks, chronological age, birth weight, and birth length were recorded per the medical chart. Body length and body weight were measured using an infantometer and weighing scales. The head circumference was measured by applying a plastic tape around the forehead (above the eyebrows) and the occipital protuberance. These measurements were converted to percentiles according to the growth charts for Taiwanese children released by the Ministry of Health and Welfare. The diet was assessed using a semi-quantitative frequency method, and the included food items were tofu, chicken, pork, beef, fish, egg yolk, viscera, vegetables, rice, wheat, baby rice cereal, baby wheat cereal, formula milk, juice, and puree. Complementary food and formula milk intakes were quantified based on standard bowls, spoons, and feeding bottles. Iron intake from the complementary food was calculated using the Nutritional Chamberlain Line, Nutritionist Edition, version 2002 (E-Kitchen Business Corp, Taiwan). Iron intake from formula milk and baby cereal was calculated according to their nutrient labels. Iron content in breast milk was obtained through actual measurement. Breast milk intake was obtained from mothers' reports. If the mothers were directly breastfeeding their infants, the total volume of breast milk intake was assessed according to the report of Lyu et al. [17]. Pre-pregnancy body weight (kg), gestational weight gain (kg), current weight, and education level were obtained from self-reported data. Current body weight (kg) and height (cm) were measured using an electronic health scale (Tanita corp., Tokyo, Japan). Complete blood count of the mothers, including Hb level, hematocrit (Hct) level, and mean corpuscular volume (MCV) before delivery at 37–40 weeks pregnancy were recorded according to the medical charts.

#### *2.3. Breast Milk and Blood Collection*

Breast milk from the mothers' unelated breast was collected using an electric milking machine (Lactina, Medela, Switzerland) for 20 min, which was then mixed, dispensed, and ice-cooled at −80 ◦C until analysis. Blood samples (4 mL) of infants were collected through an arterial puncture of one arm into vacutainers with and without anticoagulant ethylenediaminetetra-acetic acid (EDTA) by a trained technician. Serum was collected after centrifugation at 1400× *g* for 10 min at 4 ◦C and immediately sent to Central Laboratory, Shin-Kong Wu Ho-Su Memorial Hospital, for analysis.

#### *2.4. Biochemical Analyses*

Complete blood count was determined using an automatic blood cell analyzer (Biotecnica Instruments SpA, Roma, Italy). Ferritin was detected using a chemiluminescent immunoassay (Roche Diagnostics, Lewes, UK). Serum iron was analyzed using the ferrozine method (Siemens Healthcare, Marburg, Germany). Total iron binding capacity (TIBC) is the ability of transferrin to bind with iron that was measured by chemistry analyzer using dedicated reagents (Siemens Healthcare, Marburg, Germany). Transferrin saturation (TS, %) represents the percentage of transferrin bound to iron ions, calculated by dividing serum iron concentration by TIBC and multiplying the result by 100.

#### *2.5. Breast Milk Iron Content Analysis*

Aliquots of 0.5 mL of the mixed breast milk sample was added to 1.5 mL of 70% nitric acid and 0.5 mL of 30% hydrogen peroxide, separately. After mixing and allowing the samples resting for one night, the mixture was digested in a 50 mL polypropylene digestion bottle at 95 ◦C for 1 h. After cooling at room temperature, the digested sample was diluted using 50 mL of deionized water. Subsequently, 1 mL of the dilution was pipetted into a 15 mL centrifuge tube and diluted with 10 mL of 2% aqueous nitric acid solution to detect the iron content by using inductively coupled plasma mass spectrometry (ICP-MS) (ThermoFisher Scientific, Bremen, Germany) [18]. The iron content in breast milk was calculated using a standard curve constructed using pure iron standards for ICP-MS (Merck, Darmstadt, Germany); with an *R* value of ≥0.99, coefficient of variation (CV) at 2%, and a recovery rate of 80–120%.

#### *2.6. Statistical Analysis*

The WHO defines ID as a serum ferritin level less than 15.0 ng/mL and IDA as serum ferritin level less than 15.0 ng/mL and Hb less than 10.5 g/dL. We divided the subjects into three groups according to the WHO definitions: The normal, ID, and IDA groups. All data were confirmed to have a normal distribution by using the Kolmogorov–Smirnov test. Data are presented as means ± standard deviations (SDs), median (interquartile range), or percentage. Intergroup differences were determined using one-way ANOVA, followed by the Scheffé method for post hoc test or nonparametric statistics. Pearson's chi-squared test was used to assess categorical variables. The correlation between Hb and dietary iron intake was determined using the Pearson correlation test. The association between feeding types and anemia was determined using multivariable logistic regression. All data analyses were performed using SPSS (version 19; SPSS Inc., Chicago, IL, USA). Differences were considered significant at *p* < 0.05.

#### **3. Results**

#### *3.1. Participant Characteristics and Infant Anemia Diagnosis*

A total of 1368 infants were eligible for this study. However, 779 mothers did not provide consent to extract their infants' blood, and therefore, the 589 subjects were ultimately enrolled in this study. However, blood draws were unsuccessful in 39 infants. Thus, a total of 550 cases were included for data analysis. Because no introduction of complementary food to infants over six months of age was considered as abnormal feeding, four infants aged eight months and two aged 12 months were excluded accordingly. In addition, 35 infants with a WBC count of >0.000/mm3, suspected of infection, were excluded. Data on the iron status analysis of 509 infants are presented in Figure 1.

Anemia was defined according to the WHO criteria, and 49 (10%) and 21 infants (4%) were diagnosed with ID and IDA, respectively, by the physician (Table 1). No statistically significant intergroup differences were noted in terms of sex gestational age, birthweight, body length, body weight, and head circumference of infants. The formula-fed type (52.8%) was the predominant population in the healthy group. The breastfed infants had a significantly higher prevalence of ID (65.3%) and IDA (85.7%) than the mixed-fed and formula-fed infants (*p* < 0.001). No significant intergroup differences were observed in terms of the mother's age, body mass index (BMI), gestational weight gain, and education.


**Figure 1.** Flowchart of the enrolment of infants.

**Table 1.** Demographic characteristics of subjects diagnosed with iron deficiency (ID) or iron deficiency anemia (IDA) in the first year of life a.


<sup>a</sup> Values are expressed as mean ± SD or *n* (%); <sup>b</sup> According to definitions by the World Health Organization, iron deficiency (ID): Serum ferritin < 15.0 ng/mL, iron deficiency anemia (IDA): Serum ferritin < 15.0 ng/mL and hemoglobin < 10.5 g/dL.; <sup>c</sup> Definition of feeding type: Breast-fed means that the dairy products in the infant's diet are all breast milk; Mix-fed means that the dairy products in the infant's diet include breast milk and formula milk; Formula-fed refers to the dairy products in the infant's diet only formula milk. \* Differences between groups were tested using one-way ANOVA, followed by the Scheffé method for a post hoc or Chi-square test; *p* < 0.05 was considered statistically significant.

#### *3.2. Analysis of Iron Status*

The iron status of the infants and mothers before delivery are presented in Table 2. Biochemical parameters, ferritin, and TS were significantly low in ID and IDA groups. Although the mothers of infants with IDA had a slightly lower Hb level before delivery, no statistical difference was observed.


**Table 2.** Hematologic data of subjects a.

<sup>a</sup> Data are presented as mean ± SD or median (interquartile range); <sup>b</sup> ID, iron deficiency; IDA, iron deficiency anemia; Hb, hemoglobin; TS, transferrin saturation; Hct, hematocrit; MCV, mean corpuscular volume; <sup>c</sup> Means in the column with different superscripts indicate a significant difference (*p* < 0.05), tested using one-way ANOVA, followed by the Scheffé method for post hoc test or the Kruskal–Wallis test.

#### *3.3. Iron Intake of Infants*

The total iron intake was calculated as the sum of the iron contents in milk and complementary food, as presented in Table 3. Because complementary food was introduced in only 17 infants, and most of the complementary foods were cereals and fruit purees for infants aged 4–6 months, data of the iron intake from complementary food are not shown. During ages 1–6 months, the daily iron intake from milk in the normal, ID, and IDA groups were 3.43, 0.13, and 0.13 mg, respectively. Moreover, the normal group had a higher total iron intake (3.49 mg daily) than the ID (0.13 mg daily) and IDA (0.13 mg daily) groups at ages 1–6 months. The low total iron intake in infants of the ID and IDA groups could be attributed to exclusive breastfeeding, and both ID and IDA were observed in infants aged 4–6 months.

**Table 3.** Iron intake of infants aged 1–6 months and 7–12 months a.


<sup>a</sup> Data are presented as median (interquartile range); <sup>b</sup> ID, iron deficiency; IDA, iron deficiency anemia; <sup>c</sup> Iron intake from milk was the sum of iron intake from formula milk and breast milk.; <sup>d</sup> Total iron intake was the sum of iron intake from milk and complementary food; \* Intergroup differences were tested using the Kruskal–Wallis test or chi square test; *p* < 0.05 was considered statistically significant.

The normal group had the highest iron intake from milk (5.04 mg daily), whereas, the ID and IDA groups had a low iron intake from milk. This could be attributed to the continuous breastfeeding of the infants in the ID and IDA groups. No intergroup differences were noted in terms of the iron intake from complementary food. Therefore, milk was the major source of iron intake in infants aged 7–12 months. Furthermore, we determined that the prevalence of ID and IDA in infants aged 7–12 months was 20.4% and 6.6%, respectively, which was significantly higher than 3.7% and 2.7%, respectively, in infants aged 1–6 months. Our results revealed that infants with ID or IDA before six months were all in aged 4–6 months and breastfed. The medium iron intake in infants aged 4–6 months in the normal, ID and IDA groups were 5.13 mg (6.54), 0.13 mg (0.18), and 0.13 mg (0.29), respectively. In aged 7–12 months, infants with IDA had the highest breastfeeding rate, followed by those with ID.

Iron is essential for hematopoiesis. We observed that the total dietary iron intake was positively correlated with Hb (*r* = 0.292, *p* < 0.001) (Figure 2A). We further divided the data into the following two subgroups: One to six months (Figure 2B) and 7–12 months (Figure 2C). The data revealed that the correlation between total dietary iron intake and Hb in infants aged 7–12 months was higher than in those aged 1–6 months (*r* = 0.390, *p* < 0.001 vs. *r* = 0.130, *p* = 0.028).

**Figure 2.** Correlations between total dietary iron intake and hemoglobin at 1–12 months (**A**), 1–6 months (**B**) and 7–12 months (**C**).

#### *3.4. Association between Feeding Type and Iron Status*

Logistic regression models were employed to identify predictors of ID and IDA. Our data revealed that feeding type was a major indicator in predicting anemia (Table 4). In model 1, no covariates were adjusted, and in model 2, gestational week, birthweight, sex, body weight percentile, body length percentile, and age of infants were adjusted. In model 3, in addition to the aforementioned variables for infants, mothers' BMI, gestational weight gain, education level, and Hb concentration before delivery were also adjusted. Compared with formula-fed infants, breastfed infants had a higher odds ratio (OR) for ID (OR: 2.715; 95% CI: 1.830–4.030) and IDA (OR: 4.338; 95% CI: 2.108–8.927) in model 1 (*p* < 0.001). Furthermore, ORs for ID and IDA significantly differed after adjustment for variables in models 2 and 3.


**Table 4.** Association between feeding type and iron status.

<sup>a</sup> SE, standard error of mean; OR, odds ratio; 95% CI, 95% confidence interval; <sup>b</sup> Model 1: Not adjusted; Model 2: Adjusted for gestational week, birthweight, sex, body weight percentile, body length percentile, age; Model 3: Adjusted for gestational week, birthweight, sex, body weight percentile, body length percentile, age of infants, as well as mothers' BMI, gestational weight gain, education level, and hemoglobin level before delivery; \* *p* < 0.05.

#### **4. Discussion**

The period of infancy constitutes a critical window of growth and brain development, and thus, micronutrient deficiencies during this period may have adverse effects on neurocognitive functions. Iron deficiency is the most prevalent nutritional deficiency among infants in developing countries. Women of childbearing age are at risk of iron deficiency because of poor iron content in the diet, increased demand for iron during pregnancy, and iron loss during menstruation and childbirth. In addition, breastfed infants are vulnerable to developing ID because of rapid growth, depletion of their iron endowment, and low iron content in breast milk and in some complementary foods. In our study, among the 509 mother–infant dyads who submitted complete data, 3.7% and 2.7% of the infants aged below six months had ID and IDA, respectively. However, in infants aged above six months, the prevalence of ID and IDA rapidly increased to 20.4% and 6.6%, respectively. Analysis of the maternal hematologic data revealed that anemia was not detected in the mothers during the routine prenatal examination. The basic characteristics of all infants (Normal, ID, IDA) were similar except for the chronological age and feeding type.

The prevalence of ID and IDA varies greatly among countries worldwide. In India, the prevalence of ID in infants aged 3, 4, and 5 months was 5.4%, 21.4%, and 36.4%, respectively, whereas, that of IDA was 4.6%, 16.7%, and 11.4%, respectively [19]. In Turkey, Germany, and Brazil, the prevalence of ID in infants aged four months were 19.8%, 6%, and 5.7%, the prevalence of IDA in those infants were 9.5%, 0%, and 3.4%, respectively [20–22]. The prevalence of ID and IDA in infants before six months of age in our study was concordant with that observed in Germany and Brazil. In Spain, the prevalence of ID and IDA was 9.6% and 4.3%, respectively, in 12-month-old infants [23]. The prevalence of ID was 14.0%, and that of IDA was 9.4% in infants aged 9–12 months in Estonia [24]. In Saudi Arabia, out of the 274 infants aged 6–24 months studied, 126 (51%) were diagnosed as having IDA [25]. Among 619 Korean infants aged 8–15 months old, ID and IDA were diagnosed in 174 (28.1%) and 87 infants (14.0%), respectively [26]. The prevalence of ID and IDA in two cohorts of infants aged nine months old in China was 2.8% and 20.7% and 12.0% and 31.2%, respectively [27]. The prevalence of ID and IDA in infants above six months of age in our study was concordant with that observed in China and Korea.

Breast milk is considered the best food for infants, because it is highly nutritious for infant growth and contains maternal antibodies that provide defense against pathogens. A recent study indicated that human breast milk has very little iron (0.5 mg/L at one month and 0.29 mg/L at 3–5 months) [28]. Our data revealed that the average iron content of breast milk from the mothers of the infants was 0.21 ± 0.06 mg/L. Although iron in human breast milk has higher bioavailability, it may not be sufficient for infants. Therefore, infants' body iron stores meet most requirements for breast-fed infants during the first six months of life. A study concluded that a normal, healthy, full-term infant has sufficient amounts of iron until approximately 4–6 months of age [29]. Our results revealed that the prevalence of ID and IDA in infants aged 1–6 months was 3.7% and 2.7%, respectively, and these infants were all in aged 4–6 month and breastfed. Hence, breastfeeding for more than four months can slightly increase the risk of ID and IDA with lower iron stores.

Studies have indicated that the risk factors for the development of ID included small-for-gestationalage, infants below 10th percentile weight for gestation, infants of diabetic mothers, very-low-birth weight preterm neonates (VLBW, <1500 g at birth) and infants with lower iron stores [30,31]. Therefore, although the iron status of our subjects seems to be more related to iron intake, studies have shown the smoking, obesity, and childbirth by caesarean section also affected the iron status of infants [32]. These factors were not considered in the present study. In the absence of inflammation, serum ferritin measurement is the most specific test to determine total iron content stored in the body. Our data revealed that infants in ID and IDA groups with lower serum ferritin levels reflected depleted iron stores. Another indicator of iron deficiency was TS. In cases of iron deficiency, serum iron is reduced, and TIBC is increased, resulting in a substantial reduction in TS. A threshold of 16% is generally used

to screen for iron deficiency. Our data revealed that subjects with ID and IDA had a TS below the threshold and TS in IDA group was lower than that in ID group.

Another possible reason causing iron depletion in infants below six months of age could be maternal iron deficiency during pregnancy, which results in offspring with inadequate iron storage in the body. Some studies have found little or no correlation between maternal and neonatal iron status, whereas, others have suggested that the fetus was vulnerable to maternal ID [33,34]. Mothers of IDA infants had lower Hb level, although there were no statistical differences. We further analyzed the correction between infants Hb level and mothers Hb level, and the results were not correlated. Although the mother's iron nutrition status during pregnancy may affect the child's iron nutrition status, our results revealed that feeding still mainly affects the infant's iron nutrition status. In addition, the timing of umbilical cord clamping affects iron stores in the newborn. A study indicated that delay in cord clamping increases the red blood cell volume and iron stores in infants [35]. Hence, cord clamping could be a feasible solution to improve the iron status of infants.

The WHO recommends exclusive breastfeeding of infants for the first six months of life. Thereafter, infants should receive complementary foods with continued breastfeeding up to two years of age or above. Taiwan's health policy also follows this recommendation. In this study, among 328 infants below six months of age, only 17 were introduced to complementary food. Most of these foods were cereals or fruit juices that had low iron content. Thus, in addition to prenatal storage, formula milk contributed more iron to the infants below six months of age. Although human milk has a high bioavailability of iron, the iron content was so low that exclusive breastfeeding increased the risk of ID and IDA. We determined that the total iron intake was positively correlated with Hb level in infants below six months of age, thereby indicating that although prenatal storage was crucial to maintain iron status, additional dietary iron intake can improve the Hb levels and prevent ID and IDA, especially in infants aged 4–6 months. Evidence from randomized control trials suggests that the rate of IDA in breastfed infants could be positively altered by the introduction of solids at four months of age [36]. The American Academy of Pediatrics recommends that exclusively breastfed, full-term infants receive 1 mg/kg of iron supplements per day from the age of four months [37]. Taiwan's maternal and child policy particularly emphasizes the benefits of breastfeeding. The exclusive breastfeeding rate for infants at six months in Taiwan increased from 20.1% in 2004 to 50.2% in 2011 [38]. Therefore, introducing complementary food early or providing iron supplementation is imperative for infants aged 4–6 months who are exclusively breastfed.

Although infants aged 7–12 months were provided complementary foods, the iron intake from complementary foods was not different among the normal, ID, and IDA groups. Notably, milk was the major source of iron. Upon further analysis of data, we observed that up to 70% of infants with ID and 90% of infants with IDA were still breastfed. This result indicated that infants who are still breastfed for more than six months do not obtain adequate iron from breast milk. This suggests that mothers who still breastfeed after six months could provide high-quality iron-rich foods to reduce the prevalence of ID and IDA.

When we reported the iron status of infants to their mothers, most of the mothers presented their infants had good growth and not aware of the insufficient iron intake and that it increased the risk of ID or IDA. Conversely, infants who used commercial formulas instead of breast milk had better iron status because most commercial formulas are iron-fortified. Future health policies should educate mothers to prepare high-quality iron-rich foods or provide iron supplementation for breastfed infants.

The maximum iron requirement for infants aged 4–12 months is approximately 1 mg/kg or 10 mg/day. The average full-term infant requires 8 mg of iron daily from approximately six months of age [39]. Our data demonstrated that the median iron intake of infants aged 1–6 months was 3.49 mg/day, and the median iron intake of infants aged 7–12 months was 6.47 mg/day, which could reach the normal iron nutritional status. Infants with IDA had normal growth and no obvious symptoms except for pallor were observed, and no infants were suspected of having anemia by caregivers. Therefore, the presentation of iron deficiency is subtle and can be detected accurately only by medical personnel.

Although our results determined that breastfeeding was associated with an increased the prevalence of ID and/or IDA, the advantages of breast milk are recognized and unquestionable. Thus, more attention should be paid to the problem of iron deficiency in infants, and strategies should be proposed, including improving maternal iron status, introducing high-quality complementary food early, and even providing iron supplement to breastfed infants. The Pediatrics Association of Taiwan revised the guidelines for breastfed infants in 2016. The content includes the following: (1) Encourage full-term infants to start breastfeeding as soon as possible after birth, (2) Continue to breastfeed until one year of age. After one year of age, the mother may continue to breastfeed the infants, (3) Breastfed infants should be started on complementary foods at 4–6 months of age. If no complementary foods are added after four months, oral iron supplementation should be started at 1 mg/kg/day. Iron deficiency affects the development of cognition, nerves, and behavior, some of which are long-term and irreversible [40]. In this study, all infants with ID and IDA improved iron status and anemia after medical iron supplementation and regular checkups by pediatricians.

The strength of the present work is that it is the first to look at the association between breastfeeding and ID and IDA during infancy in Taiwan. The results can be used as a reference for nutrition policymakers. However, the study had some limitations. First, this was a cross-sectional study that was unable to clarify the effects of longitudinal nutritional status on the development of ID and IDA. Second, the sample size was relatively small, and all participants lived in northern Taiwan, which might limit the generalizability of the results. Therefore, additional large-sample, multicenter studies are required. Third, we only prioritized examining several clinical iron markers that are more likely related to iron status. Fourth, the lack of information regarding the initiation of complementary feeding, that could significantly impact iron status in infants. Nevertheless, further studies are warranted, including those for following up infants with iron deficiency in the first year to analyze whether it affects their cognition function and growth, as well as whether early introduction of high-quality iron-rich complementary foods can improve the iron status of infants.

#### **5. Conclusions**

Our data revealed that exclusive breastfeeding increases the prevalence of ID and IDA in infants aged 4–6 months, and prolonged breastfeed above six months can significantly increase the prevalence of ID and IDA. Although iron in breast milk has good bioavailability, its low iron content results in insufficient iron intake in infants and increases the risk of anemia. Hence, health policies should encourage the early introduction of iron-rich complementary foods and educate mothers to prepare high-quality complementary foods to reduce the risk of anemia, especially in infants above six months. In addition, if no complementary foods are added after four months, oral iron supplementation should be started in breastfeeding infants.

**Author Contributions:** Conceptualization, S.-C.L. and C.-M.C.; methodology: All; data curation: S.-C.M., Y.-L.C., L.-Y.T., Y.-T.K., I.-M.C. and Y.-C.C.; validation: S.-C.M., C.-K.S. and M.-L.C.; drafting of the manuscript: S.-C.L.; critical revision of the manuscript: C.-M.C. and S.-C.M.; final approval of the version to be published: S.-C.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the Ministry of Health and Welfare (DOH101-HP-4204 and DOH102-HP-4203).

**Acknowledgments:** We thank the participants and the medical staff for their participation. This manuscript was edited by Wallace Academic Editing.

**Conflicts of Interest:** The authors have no conflicts of interest to declare.

#### **References**


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*Article*
