At the beginning of the study, except for lower ferritin concentration (between 13 and 25 ng·mL-1) and increased transferrin concentration in the MID group, similar hematological values were found in the MID and C groups (Table 2). In the MID group, [Hb] ranged from 12.2 to 14.0 g·dL-1 and one subject showed an MCV below the normal range (78 fL).
During the iron supplementation period, two SID subjects reached the criteria to stop the supplementation (ferritin >=25 ng·mL-1 and a plateau in Hbmass) after 10 wk, five subjects finished after 14 wk, and one subject met the criteria after 18 wk. At the end of the iron supplementation period, [Hb], ferritin concentration (Fig. 2a and b), and all other markers of the iron status were normalized in this group and did not differ from the values in the C group (Table 2). Twelve weeks after the supplementation, the ferritin level was markedly reduced again and the transferrin was increased whereas all of the other parameters remained constant (Table 2).
In the MID group, the ferritin (Fig. 2b) and transferrin concentrations were shifted into the normal range during the supplementation period and [Hb] (Fig. 2a), MCH, and MCHC exhibited slight but significant increase (Table 2). No change was observed during the postsupplementation period.
In the SID group, Hbmass significantly increased until the end of the supplementation period by 15.6% ± 11.0% (77.0 ± 37.7 g) (Fig. 2c) and reached the level of the C group. Hbmass remained at this level for the following 12 wk. In the MID group, Hbmass slightly increased (2.2% ± 3.7% or 16.1 ± 18.0 g, with P < 0.05) and remained at this elevated level for the following weeks (Table 2). Compared with the C group, Hbmass in the MID group demonstrated significant increase after the supplementation period (P < 0.05) and tendency toward higher values after 12 wk (P = 0.07). The considerable variability in the change of Hbmass within the groups (Fig. 2c) indicates the very different individual responses during and after the iron supplementation, as presented in Figure 2d and e. The individual change in Hbmass after the iron supplementation period ranged from 34.8 (6.0%) to 155.9 g (39.0%) in the SID group and from -5.2 (-1.0%) to 52.5 g (10.2%) in the MID group.
The BV exhibited slight increase in the SID group, and this increase was significant compared with the level in the C group (P < 0.05). The erythrocyte volume increased in a manner similar to that of Hbmass, whereas PV remained unchanged over the entire observation period (Table 2).
To demonstrate the dependence of the individual Hbmass response on the baseline ferritin value, a regression analysis was performed (Fig. 3a). At ferritin levels between 10 and 25 ng·mL-1, a moderate increase in Hbmass was found. If the baseline ferritin values were less than 10 ng·mL-1, an exponential increase in Hbmass was found and the lowest ferritin value (3 ng·mL-1) was related to the greatest response (156 g). Significant but weaker relations were demonstrated for the Hbmass response versus the initial [Hb] (r = 0.60; P < 0.01) and initial MCH (r = 0.52; P < 0.05), and a nonsignificant relation was observed with the initial MCV (r = 0.38; not significant).
Changes in Hbmass and changes in [Hb] were moderately correlated (r = 0.67, P < 0.01) (Fig. 3b); closer relations were found for changes in Hbmass with changes in MCV (r = 0.90, P < 0.001) (Fig. 3c) and with changes in MCH (r = 0.87, P < 0.001). Regression analyses of changes in [Hb] versus changes in MCV (r = 0.69) and changes in MCH (r = 0.71) also showed significant (in both cases, P < 0.01) but weaker relations when compared with those for Hbmass. The normal variability of Hbmass and [Hb] during the observation period expressed as the coefficient of variation of the control group was 2.25% for Hbmass and 3.10% for [Hb].
In the SID group, 1715 mg of iron was stored in hemoglobin at the beginning of the intervention, and this value significantly increased to 1975 mg after the iron supplementation period. By calculating the amount of additional iron that was stored in both Hbmass and ferritin during the supplementation period, it is possible to estimate the quantity of iron absorbed from the supplements. Although both treatment groups replenished their ferritin stores during the intervention (SID, +235 ± 105 mg; MID, +202 ± 149 mg iron), the SID group also stored approximately the same amount of iron in hemoglobin. In total, the SID group incorporated 495 ± 205 mg of iron in both the ferritin and hemoglobin stores; in contrast, the amount of iron incorporated was 234 ± 141 mg in the MID group and -43 ± 148 mg in the C group (Fig. 4).
The V·O2max value was slightly greater than its initial value in the SID group after the supplementation period, whereas no changes occurred in the MID and C groups (Table 2). There was a significant relation between the changes in Hbmass and the changes in V·O2max (y = 2.3 x – 4.3; r = 0.65; P <= 0.01), thereby indicating that a change of 1 g in Hbmass caused a change in V·O2max of 2.3 mL·min-1 (Fig. 3d).
This study describes the time course of the recovery of Hbmass in severely and moderately iron-deficient recreational athletes during oral iron supplementation period. The main findings are the very individual responses in the magnitude and time course of Hb production in the SID group (in the range of 35–156 g over 10–18 wk) and in the MID group (a range of -5 to 52 g over 10 wk). Close relations between the Hbmass response and MCV and MCH were found. The changes in the main iron stores (i.e., ferritin and hemoglobin) were quantified, as follows: 495 mg in the SID group and 230 mg in the MID group.
In a case study of a severely iron-deficient athlete, Hbmass was monitored to evaluate the effect of iron supplementation via i.m. iron injections combined with oral iron supplementation and Hbmass increased by 77% within 7 wk (13). In the case of an athlete with normal iron status, however, i.m. injections failed to have any effect (1). In another study, i.v. injections of iron in iron-deficient, nonanemic elite athletes yielded 5% increase in Hbmass within 8 wk whereas no effect was observed in a similar group during oral iron administration (14). Similar findings of no changes in Hbmass were reported by Friedmann et al. (12), who monitored iron-deficient, nonanemic elite junior athletes (ferritin, 16 μg·L-1; [Hb], 13.8 g·dL-1) during a 12-wk iron supplementation period (100 mg of Fe twice per day). To date, no study has monitored the effects of oral iron supplementation on Hbmass in severely iron-deficient subjects.
In this study, iron supplementation was a successful treatment for the initially iron-deficient subjects in both groups and led to normalization of all iron status markers. Even after 2 wk of supplementation, all of the SID subjects exhibited increase in Hbmass, which ranged between +2% (11 g) and +22% (76 g) and was followed by very individual further elevation of between 6% and 39%. This quick response at the beginning of oral iron supplementation is described in the literature (47) but has not been precisely quantified.
Only two of the eight subjects in the SID group met the criteria (ferritin >=25 ng·mL-1 and increase in Hbmass <20 g within the previous 3–4 wk) to stop supplementation after 10 wk, thus indicating that 10 wk was insufficient in most cases for complete recovery from severe iron deficiency. This time evolution of recovery is consistent with current guidelines, which recommend at least 3 months of oral iron supplementation (31). In contrast with the subjects studied by Friedmann et al. (12) and Garvican et al. (14), the MID subjects also exhibited small but significant mean increase in Hbmass. In two of the 14 subjects, the increase in Hbmass reached approximately 10%, which is far greater than the mean response to physiological stimuli, e.g., training at sea level (38) or even at altitude (19,43). The fact that MCV and MCH increased by 3 fL and 2 pg, respectively, in both of these subjects emphasizes recovery from iron deficiency.
The changes in Hbmass in response to iron supplementation differed among individuals and were dependent on the severity of iron deficiency, which was indicated by the initial ferritin value (Fig. 3a). In this study, relatively small responses in Hbmass were observed for initial ferritin values between 10 and 25 ng·mL-1. At lower values, however, exponential increase occurred. These data quantify the effects that occur during iron supplementation when the threshold for severe iron deficiency of 10–12 ng·mL-1 (5,20,32) is passed. In contrast, four of the eight subjects with ferritin concentration less than 10 ng·mL-1 had initial [Hb] that was slightly greater than 12.0 g·dL-1. In clinical practice, [Hb] thresholds are—in combination with ferritin—used to classify the degree of iron deficiency; for example, the World Health Organization (46) uses thresholds of 12.0 g·dL-1 for females and 13.0 g·dL-1 for males. The individual normal [Hb], however, exhibits considerable interindividual oscillations, ranging between 12.0 and 16.0 g·dL-1 in women and 13.5 and 17.5 g·dL-1 in men (25). Thus, a subject with a high normal [Hb] that decreases [Hb] because of iron deficiency (e.g., [Hb] decreases from 14.5 to 12.5 g·dL-1) is not classified as an anemic subject but may benefit from iron supplementation, which is particularly important for elite athletes. In this study, one female subject exhibited low ferritin concentration of 14 ng·mL-1 and normal [Hb] of 14.0 g·dL-1 and was therefore classified as nonanemic. To fall below the anemic threshold, she had to lose approximately 90 g of hemoglobin. During the supplementation period, however, she responded with increase of 8.3% (52.7 g) in Hbmass, proving to be anemic “for her” at the beginning of the study.
As described previously, in addition to limitations in the exact diagnosis of iron deficiency anemia using [Hb], the time course and magnitude of recovery after iron supplementation may also not be precisely quantified by [Hb]. As demonstrated in Figure 3b, significant relation existed between Hbmass and [Hb], although this relation was due only to the data of the SID group, as no relation existed within the MID group. Because the relations between the changes in Hbmass versus MCV and MCH were stronger than those for [Hb] versus MCV and MCH, we suggest Hbmass as a more sensitive parameter to monitor recovery from iron deficiency. This opinion is supported by the lower coefficient of variation for Hbmass than that for [Hb] in the control group, indicating lower biological and/or methodological variability for Hbmass.
RCV and red cell hemoglobin content.
In the SID group, the percentage increase in RCV (10.0%) was less than the increase in Hbmass (15.2%). This difference was due to the increased incorporation of hemoglobin into premature erythrocytes (+11.1%, MCH from 27.8 to 30.9 pg), which exceeded the increase in MCV (+5.2%, from 84.5 to 88.9 fL); both effects are normal responses that occur when iron stores refill. Because PV did not change, BV was only slightly affected. Because of the increased erythrocyte count, the augmented red cell hemoglobin content and the almost unchanged BV the hemoglobin concentration increased in the SID group and also in the MID group, but to a much lower degree.
Twelve weeks after the supplementation period, mean Hbmass remained constant in the two groups whereas the ferritin concentration significantly decreased in the SID group (Table 2). This indicates that in early iron deficiency, iron stores are depleted before erythropoietic activity is affected. As demonstrated previously, severe interference with erythropoiesis occurs with very low ferritin levels. This is demonstrated by the subject who lost the greatest amount of Hbmass (approximately 50 g), which was accompanied by reduction in ferritin to 4 ng·mL-1.
Knowing the absolute Hbmass of a subject and the change in Hbmass during the iron supplementation period allows for exact quantification of the iron that is incorporated into hemoglobin molecules and, therefore, the success of iron therapy. A previous study (45) reported that the amount of nonhemoglobin body iron can be estimated using the equivalence of 1 ng·mL-1 of ferritin and 8 mg of iron. Consistent with Cook et al. (8), who found 309 ± 346 mg of iron in body stores in females, we found a very similar mean value for all three groups (315 ± 125 mg) at the end of the supplementation period. The total iron content can be calculated by accounting for the storage of 10% of the iron content in myoglobin and the involvement of 2% of the iron content in electron transport, antioxidant enzymes, and DNA replication (31). At the end of the supplementation period, the total iron content did not differ between the groups. The mean of all three groups was 2.6 g; 76% (1930 mg) was stored in hemoglobin, and 12% (315 mg) was stored in ferritin. These results closely coincide with previously reported values (29).
In the SID group, the increase in ferritin concentration paralleled the change in Hbmass, thus indicating similar time evolution for filling the two main iron stores of the body. Neglecting the baseline iron absorption rate of 1–3 mg·d-1 (36), which is counterbalanced by a similar daily excretion rate, approximately 210 mg of additional iron was resorbed from the supplemented iron dose during the first 2 wk and was equally distributed between the ferritin (95 mg) and hemoglobin (115 mg) stores. The amount of additional iron that was resorbed during the entire supplementation period was approximately 500 mg, and the amounts of that iron distributed to ferritin and hemoglobin were 235 mg and 260 mg, respectively (Fig. 4).
The amount of incorporated iron in the MID group (230 mg) was approximately 50% less than that in the SID group and was primarily used to fill the ferritin store (+200 mg) rather than for incorporation into hemoglobin. Although no previous data regarding iron incorporation into the hemoglobin of MID subjects are available, the increase in the ferritin iron store in this study is consistent with data from the literature, in which an 88- to 240-mg increase in ferritin iron is reported (31).
Although it is well known that iron deficiency anemia impairs endurance exercise performance (e.g., ref. 3), the effect of iron supplementation on V·O2max and performance, particularly for borderline iron deficiency, is debated upon. Some studies have demonstrated significant increase in V·O2max (12,26,27) or endurance exercise performance (35) after oral iron supplementation, and some studies have failed to demonstrate such an effect on V·O2max (10,24) or performance (22,30,32,33). Furthermore, in this study, we did not detect any increase in V·O2max in the MID group. Similarly, in a very recent study, Garvican et al. (14) did not find any change in V·O2max or performance in nonanemic athletes after oral iron supplementation but did find significant improvement in a similar group after i.v. iron injection (an increase in V·O2max of 1.9%); thus, they concluded that i.v. supplementation was the superior therapy.
In the SID group, however, V·O2max clearly increased by 7.4%, which is consistent with previous data (39) and was closely related to the elevated Hbmass. The slope of the regression line indicates that a change in Hbmass by 1 g as a result of the iron supplementation is associated with an increase of 2.3 mL·min-1 in V·O2max. This number is less than a previously reported value (39) for elite athletes (4 mL·min-1). This difference might result from the subjects in our study being recreational athletes, for whom factors other than Hbmass, e.g., mitochondrial oxidative capacity, may be the primary limiting factors for V·O2max (44). The two subjects with the highest training level also showed the greatest response in V·O2max related to 1 g of Hbmass (5.9 and 5.2 mL·min-1). We therefore suggest that the recovery in Hbmass resulting from iron supplementation has a high effect on aerobic capacity, particularly in well-trained endurance athletes.
Possible limitations of the study.
One limitation of the study is that menstrual blood loss was not considered in our calculations. However, the occurrence and intensity of menstruation were documented by questionnaires and did not obviously affect the data. The iron loss during normal menstruation is 0.5–1 mg·d-1 (11,28), and the mean iron incorporation in the SID group during the supplementation period was 8 mg; therefore, the effect of menstruation did not severely interfere with iron balance.
The dietary intake of iron was not controlled. The test subjects were advised to maintain their usual diet; therefore, we did not expect a significant dietary effect on our results.
Hbmass is a very sensitive tool for monitoring the magnitude of iron deficiency anemia and the time evolution of recovery during and after iron supplementation periods. Because [Hb] is acutely and chronically influenced by changes in PV, small changes in Hbmass may not be detected. We therefore recommend that regular measurements of Hbmass be used in addition to [Hb] and ferritin measurements for the early detection of iron deficiency, when the optimal Hbmass is known, and for monitoring the effectiveness of iron supplementation.
This project was funded by the World Anti-Doping Agency (08E09WS).
The authors declare no conflicts of interest. The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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Keywords:© 2015 American College of Sports Medicine
IRON DEFICIENCY ANEMIA; IRON STORES; ERYTHROPOIESIS; V·O2MAX; IRON (II)-GLYCINE-SULFATE