Research Article

Iron insufficiency diminishes the erythropoietic response to moderate altitude exposure

Abstract

The effects of iron stores and supplementation on erythropoietic responses to moderate altitude in endurance athletes were examined. In a retrospective study, red cell compartment volume (RCV) responses to 4 wk at 2,500 m were assessed in athletes with low (n = 9, ≤20 and ≤30 ng/mL for women and men, respectively) and normal (n = 10) serum ferritin levels ([Ferritin]) without iron supplementation. In a subsequent prospective study, the same responses were assessed in athletes (n = 26) with a protocol designed to provide sufficient iron before and during identical altitude exposure. The responses to a 4-wk training camp at sea level were assessed in another group of athletes (n = 13) as controls. RCV and maximal oxygen uptake (V̇o2max) were determined at sea level before and after intervention. In the retrospective study, athletes with low [Ferritin] did not increase RCV (27.0 ± 2.9 to 27.5 ± 3.8 mL/kg, mean ± SD, P = 0.65) or V̇o2max (60.2 ± 7.2 to 62.2 ± 7.5 mL·kg−1·min−1, P = 0.23) after 4 wk at altitude, whereas athletes with normal [Ferritin] increased both (RCV: 27.3 ± 3.1 to 29.8 ± 2.4 mL/kg, P = 0.002; V̇o2max: 62.0 ± 3.1 to 66.2 ± 3.7 mL·kg−1·min−1, P = 0.003). In the prospective study, iron supplementation normalized low [Ferritin] observed in athletes exposed to altitude (n = 14) and sea level (n = 6) before the altitude/sea-level camp and maintained [Ferritin] within normal range in all athletes during the camp. RCV and V̇o2max increased in the altitude group but remained unchanged in the sea-level group. Finally, the increase in RCV correlated with the increase in V̇o2max [(r = 0.368, 95% confidence interval (CI): 0.059–0.612, P = 0.022]. Thus, iron deficiency in athletes restrains erythropoiesis to altitude exposure and may preclude improvement in sea-level athletic performance.

NEW & NOTEWORTHY Hypoxic exposure increases iron requirements and utilization for erythropoiesis in athletes. This study clearly demonstrates that iron deficiency in athletes inhibits accelerated erythropoiesis to a sojourn to moderate high altitude and may preclude a potential improvement in sea-level athletic performance with altitude training. Iron replacement therapy before and during altitude exposure is important to maximize performance gains after altitude training in endurance athletes.

INTRODUCTION

Altitude training is routinely incorporated as a training tool to improve sea-level performance in endurance athletes (9, 15, 70, 72, 74). Although there is some debate about mechanisms, the weight of evidence suggests that increased oxygen carrying capacity of blood from an increased red cell compartment volume (RCV), total hemoglobin mass (Hb mass), and hemoglobin concentration (Hb) in response to the hypoxia at altitude is a key mechanism underlying improvement in maximal oxygen uptake (V̇o2max) and endurance performance at sea level after altitude training (10, 30, 43, 48, 62, 73, 74). However, the response to altitude training camps in athletes is highly variable (7, 10). Some mechanisms underlying this variability include an inadequate ‘dose’ of altitude [altitude achieved, duration of exposure per day, total number of days (8, 30, 42, 75)], natural versus simulated altitude exposure (28, 51, 60, 63), and concomitant illness in some athletes, which may inhibit the erythropoietic response (21, 31, 56, 70). Indeed, evidence from recent controlled trials is mounting that if there is no erythropoietic effect of an altitude training camp exposure (47, 65) or the accelerating erythropoiesis is prevented by phlebotomy (23), there is no increase in V̇o2max, which may affect its benefit to endurance performance postexposure.

Another factor that may interfere with the accelerated erythropoiesis of altitude exposure is availability of adequate iron stores (27, 32). This problem of iron deficiency preventing erythropoiesis in response to erythropoiesis stimulating agents is well known in the clinical arena, especially in patients on dialysis who routinely receive intravenous iron to allow response to erythropoietin (EPO) (39). Iron insufficiency is the most prevalent nutritional problem in the developed world (49), and it is also very prevalent among endurance athletes (11, 54). Iron is required for formation of hemoglobin, myoglobin, cytochromes, and enzymes involved in energy production. Iron deficiency leads to ineffective erythropoiesis and, when severe, anemia in athletes as well as in nonathletes at sea level (4, 46, 64). During an altitude sojourn, iron requirements and utilization are increased from the hypoxia-induced erythropoiesis (36, 61). Therefore, iron deficiency, even without anemia, might be most problematic during an altitude training camp. Indeed, monitoring iron status in athletes at altitude is highly recommended in the field of training for endurance athletes (32). Previous studies using chronic living at moderate altitude, even those with outcomes leading to varied conclusions, have used supplementary iron during altitude training (6, 20, 60, 65). However, to our knowledge, there are no prospectively obtained, peer-reviewed data documenting the effects of iron deficiency before altitude on erythropoietic response and improvement of endurance performance to altitude training camp. Additionally, optimal iron supplementation strategies for athletes before and during altitude training camp required to induce adequate erythropoiesis and training adaptation remain unknown.

To address this question, we first retrospectively analyzed the data from our initial altitude training studies [summarized in (44)] designed to compare the effects of various combinations of altitude living and training. In these studies, we found improved performance in all groups but noted several confounding factors that obscured the unique effects of altitude living. These factors included nutrition, primarily iron insufficiency, and previous training history, or ‘training camp effect’. For the purpose of the present study, we retrospectively analyzed these early data to compare erythropoietic and training responses to 4 wk of moderate altitude exposure (2,500 m) between athletes with low and normal iron stores at the beginning of training (Study I). In this retrospective analysis, we also aimed to determine the daily iron requirement for competitive athletes to induce an increase in RCV after an altitude sojourn.

In a subsequent prospective study (Study II), we report the results of a strategy designed to provide sufficient iron by oral iron supplementation before and during altitude exposure and the effects on the erythropoietic response to altitude training and sea-level athletic performance in athletes with sufficient iron stores. Although the primary physiological and exercise performance results of this prospective study have been widely reported and cited (10, 43), this manuscript focuses specifically on the role of iron deficiency/sufficiency on the erythropoietic effect of moderate altitude exposure in endurance athletes. Our hypotheses were as follows: 1) that RCV and V̇o2max would not increase after exposure in individuals with low iron stores (serum ferritin level ≤ 20 ng/mL in women and ≤ 30 ng/mL in men) before altitude and 2) that sufficient iron supplementation before and during an altitude sojourn would induce an expected increase in RCV and sea-level athletic performance in endurance athletes.

METHODS

Subjects

Nineteen well-trained distance runners (6 women, 13 men) participated in Study I [retrospective study, primary outcomes reported in Levine and Stray-Gunderson (44)]. Additionally, 39 well-trained distance runners (12 women, 27 men) participated in Study II [prospective study, primary outcomes reported in Levine and Stray-Gunderson (43)]. All of the subjects were recruited from collegiate track and cross-country teams, local running clubs, and USA Track and Field development teams. They were required to be competitive at a distance between 1,500 m and the marathon and to have a recent personal best 5,000-m time (or equivalent) of <16:30 min:s for men and <18:30 min:s for women. All subjects were born and had grown up at sea level. None had been to altitude above 1,500 m for a period exceeding 1 wk in the previous 10 mo. They had no apparent illnesses at the time of enrollment. All subjects gave their voluntary written informed consent to a protocol approved by the Institutional Review Board of the University of Texas Southwestern Medical Center.

Study Design

An outline of the study is shown in Fig. 1; it consists of two protocols, protocol I for Study I and protocol II for Study II. All testing took place in Dallas, Texas (150 m) at sea level except for three blood samplings that took place in either San Diego or Utah during the field training camp in protocol II.

Fig. 1.

Fig. 1.An outline of Study I (retrospective study) and Study II (prospective study).


Training altitude for athletes living at 2,500 m in both protocols ranged from 1,250–3,000 m. There were no significant differences in the response in RCV to living at altitude between the subgroups training at 1,250 m or 2,500–3,000 m (data not shown). Therefore, data from subjects living at the same altitude regardless of training altitudes were pooled for the primary analyses of the present study.

Protocol I.

Protocol I consisted of a 4-wk field training camp living at 2,500 m (Deer Valley, Utah) and training from 1,250–3,000 m without formal iron supplementation; 1 wk of testing was performed just before and after the training camp (see Measurements). Based on the serum ferritin level measured just before the training camp, athletes were identified as either normal or low iron stores groups (see Iron Stores), and the responses to the training camp were analyzed retrospectively.

Protocol II.

Protocol II consisted of four major phases: 2-wk sea-level lead-in phase; 4-wk sea-level training; 4-wk field training camp either living at 2,500 m and training from 1,250–3,000 m or living and training at sea level; and another 3-wk posttraining and testing at sea level. The specifics of this protocol have been described in detail previously (43) and will only be summarized briefly here and in Fig. 1. All subjects were supplemented with iron elixir titrated to serum ferritin concentration from the sea-level lead-in-phase to the end of the field training camp as described below (in Iron Maintenance or Replacement Therapy).

Sea-level lead-in phase.

Runners were brought to Dallas, 2–4 wk after the spring track season for a 2-wk period of supervised training at sea level and familiarization with laboratory testing procedures. During this phase, serum ferritin concentration was measured for the assessment of bone marrow iron stores (see Iron Stores). Iron maintenance or replacement therapy with high-dose oral liquid iron supplementation was initiated from this phase for all subjects (see Iron Maintenance or Replacement Therapy).

Sea-level training.

After the lead-in phase, runners underwent a period of supervised training and correction of iron deficiency at sea level for 4 wk (see Training Program) that was designed to provide a longitudinal sea-level control.

Field training camp.

After completion of the post sea-level training tests, the 39 subjects were then matched for sex, 5,000-m time trial performance, and training history and randomized using a stratified, balanced design to groups living either at 2,500 m (Deer Valley, Utah, n = 26) or sea level (Chula Vista, California, 150 m, n = 13) where they lived for 4 wk. Runners in the altitude living group trained from 1,250–3,000 m. Details of the living and training environments for each group were described previously (43).

Sea-level posttraining and testing.

The first week after return from the field training camp was a testing week. Blood compartment volumes and erythropoietic and hematological parameters were measured within 36 h after descent. Treadmill maximal exercise tests were performed on the fourth and fifth day after descent.

Iron Stores

Bone marrow iron stores were assessed by serum ferritin concentration, which was measured before and after the field training camp in protocol I and during the sea-level lead-in phase; before and at 2 and 4 wk of the sea-level training; at 36 h, 2 and 4 wk after the start of the field training camp (altitude or sea level); and within 36 h after descent, 2 and 3 wk after returning to sea-level posttraining in protocol II. Low iron stores were defined as a serum ferritin concentration ≤ 30 ng/mL for men (16) and ≤ 20 ng/mL for women (41). To define iron deficiency, a constant cutoff value for serum ferritin for both sexes has been used in previous studies (12, 32). However, we chose to utilize different serum ferritin iron deficiency criteria for men and women, based on well-established population norms showing lower serum ferritin values in women versus men (71).

Iron Maintenance or Replacement Therapy

In Study I, no formal iron supplementation was administered or recommended for runners during the study (Fig. 1). The initial ferritin level just before ascent was reported to all runners 1 wk after ascent, and runners were given dietary advice on sources of dietary iron. To investigate the relationship between preexposure iron stores and the responses in RCV and V̇o2max to altitude exposure, in Study I, the amount of iron intake from a source other than food was maintained throughout training for each runner. That is, any runner who was already taking iron supplements (as part of daily vitamins) was required to maintain the supplementation throughout the remaining 3 wk of the study, although we did not monitor or record iron supplementation practices for each runner. Runners who had low ferritin levels were recommended to begin oral iron supplementation after completion of the study.

In Study II, an aggressive oral liquid iron supplementation strategy was administered, beginning with the first ferritin measurement 5–6 wk before and throughout 4 wk of a training camp for both altitude and sea-level groups (Fig. 1). Iron dosage was adjusted weekly based on serum ferritin levels and subject tolerance. Runners who had initially normal ferritin levels (> 30 ng/mL for men and > 20 ng/mL for women) maintained that level with steadily increasing doses of liquid ferrous sulfate (Feosol, Meda Consumer Healthcare, Marietta, GA), from 44 mg up to 264 mg of elemental iron daily as required. Runners who had initially low ferritin levels received steadily increasing dose of liquid iron from 44 mg up to 396 mg of elemental iron daily. The oral iron supplement was administered 1–3 times/day, 30 min before a meal, with 5–15 mL of liquid iron elixir (5 mL = 44 mg of elemental iron) in 150–250 mL of orange juice with 500 mg of powdered Vitamin C. During this study protocol, staff instructed the subjects and periodically monitored ingestion of supplement. Compliance, serum ferritin levels, and any complications were discussed in weekly meetings of investigators, staff, and individual subjects. No major complications occurred during the study. Gastric upset and/or loose stools occurred in ~30% of subjects, but the symptoms were relieved by temporary dose reduction. Several athletes complained of dental staining and one required professional cleaning. This was minimized by quick consumption of supplement without holding the liquid in the mouth. Study supplementation was discontinued after the 4-wk field training camp in most subjects, although subjects who were still iron deficient after the camp were allowed to continue supplementation.

Training Program

Training was conducted according to an individualized template based on a 4-wk mesocycle intended to provide increasing volume and intensity over the first 3 wk, with a slight taper during the last week as previously described (43). The training program during the 4-wk field training camp was similar between protocols I and II and also matched the training program during the sea-level training in protocol II, on the basis of the same 4-wk mesocycle.

All training sessions were directly supervised and observed by the investigators or staff and carefully monitored. Each runner kept a detailed training logbook that included duration, intensity, and approximate distance of each workout, along with resting and training heart rate (HR) measured during every training session using a HR monitor (Polar CIC, Polar, Kempele, Finland). Diet was also monitored to ensure adequate nutrition in addition to the iron supplementation.

Dietary Assessment

Nutrient intake was assessed at the beginning and near the end of each 4-wk training camp by having the subjects fill out 3-day dietary logs. The subjects were instructed on how to fill out the logs, and staff supervised the data recording. Food was weighed on a food scale before consumption. Fluid intake was measured to the nearest 10 mL with calibrated water bottles. Food preparation was described in the logs. These data were keyed into computers by staff utilizing nutrition software (Computrition, West Hills, CA), and nutrient intake was calculated. Computer output was double-checked and verified for accuracy. Dietary iron (in milligrams of elemental iron) was averaged for the 6 days of each 4-wk training camp for each subject.

Measurements

Body composition.

Percent body fat was measured by skinfold thickness in duplicate with the equation of Siri et al. (67). Lean body mass was calculated from body weight and percent body fat.

Treadmill maximal exercise tests.

In both protocols V̇o2max was determined as previously described using an incremental test on a treadmill and confirmed with a supramaximal test the following day (35). The highest oxygen uptake (V̇o2) during either test was accepted as V̇o2max. V̇o2 was measured using the Douglas bag method, gas fractions were analyzed by mass spectrometer (Marquette MGA 1100, Marquette Electronics, Milwaukee, WI), and ventilatory volume was measured with either a Tissot spirometer or dry-gas meter (Collins, Braintree, MA). Additionally, HR was monitored continuously (Polar CIC) during the test.

Blood component volumes.

Plasma volume (PV) was measured using the Evans blue dye dilution technique (17). The runners reported to the laboratory at 7:00 AM normally hydrated but in a fasted state. After at least 30 min of quiet, supine rest, baseline blood samples were taken; then, 2.5 mL of Evans blue dye was injected through a peripheral intravenous catheter and flushed with ~8 mL of injectable saline, taking care to flush all dye out of the catheter. The exact amount of the dye injected was determined by weighing the injection syringe to the nearest 10−6 g (Mettler-Toledo, Columbus, OH) before and after injection of the dye. Venous blood was drawn at 10, 20, and 30 min after injection for the measurement of absorbance at 620 and 740 nm via spectrophotometry (DU 600, Beckman, Beckman Instruments, South Pasadena, CA). PV was calculated from these values. Blood volume (BV) was calculated from PV and hematocrit (Hct) using appropriate corrections for trapped plasma and peripheral sampling (34). Red cell compartment volume was defined as BV minus PV.

Erythropoietic and hematological parameters.

Blood samples were collected between 7:00 and 8:00 AM after at least 30 min of quiet, supine rest. Subjects had fasted except for water 12 h before phlebotomy and had had no exercise in the previous 18 h. For samples collected in Dallas, 2 mL were collected in an EDTA-2K tube for determination of complete blood count and an automated differential (Infolab Hematology Analyzer I-1800, Miami, FL). Hct was measured via microcapillary centrifuge with the same sample. Five mL of blood were collected in a serum separator tube, allowed to clot, then centrifuged (10 min at 3,000 revolutions/min) to obtain serum. The serum samples were frozen until analysis (approximately −80°C). In the field, samples collected on EDTA-2K tubes were shipped overnight (≈ 4°C) and analyzed within 48 h. A comparison study in our laboratory demonstrated no significant difference in red cell indices with up to 48 h between collection and analysis (unpublished data). The samples collected in serum separator tubes in the field were centrifuged (10 min at 3,000 revolutions/min) on site, and the serum was transferred to Nalgene cryovials and shipped overnight (≈ 4°C) to our laboratory.

Serum samples were analyzed in duplicate for iron and iron binding capacity (modified Goodwin’s methodology) by spectrophotometry (ENI Gemstar chemistry analyzer, West Caldwell, NJ). Serum ferritin and EPO levels were determined in duplicate by radioimmunoassay using commercial kits (Incstar, Stillwater, MN) and an automatic gamma counter (Cobra Autogamma, Akron, OH). The lower limit of detection for the serum ferritin kit was <1.0 ng/mL. The coefficient of variation within assay was 4.8% and 10.3% between assays for the range of values in this study. The percent cross-reactivity was <1% for human heart ferritin. The lower limit of detection for the EPO assay was <4.4 Mu/mL. The coefficient of variation within assay was 5.2% and 6.7% between assays for the range of values in this study. The percent cross-reactivity was <0.001% for all the tested substances.

Calculations

Additional iron requirements for altitude training were calculated using average data from athletes with normal initial ferritin levels in protocol I as follows. First, additional daily iron requirements for altitude training were calculated as:

[Additional daily iron requirements for altitude training]=[daily iron requirement for training athletes]+[daily iron used at altitude][daily dietary iron intake].(1)

In Eq. 1, the daily iron requirement for training athletes used was 1.9 mg/day for this group as a sex weighted average of 1.8 mg/day for men and 2.3 mg/day for women (5). Daily iron used for altitude was calculated as daily change in stored iron estimated from daily change in ferritin concentration (ng/mL) for living at altitude times 8, as 1 ng/mL of serum ferritin concentration represents 8 mg of stored iron (13, 71). Daily dietary iron intake was calculated from dietary elementary iron intake times 0.15, assuming that average elementary iron absorption is 15%, because intestinal iron absorption from the diet at sea level is ~10% (5) but can be 15–20% at 4,542 m altitude (59).

Daily change in ferritin concentration for living at altitude was calculated as change in ferritin concentration over 4 wk at altitude divided by 28 days, which was calculated as,

[Change in ferritin concentration over 4 wk at altitude]=[(ferritin mass gained by living and training at altitude for 4 wk)(ferritin mass gained by living and training at sea level for 4 wk)]/(mean PV during altitude training).(2)

In Eq. 2, ferritin mass gained by living and training at altitude or sea level was calculated from ferritin concentration and PV before and after altitude or sea-level training, respectively.

Statistics

All data values are presented as means ± SD. A Shapiro-Wilk test was utilized to determine normality of all dependent variables. To address if there was an effect of preexposure iron stores on the multiple dependent variables after a 4 wk altitude training camp without formal iron supplementation, in Study I, serum ferritin concentration, Hb, RCV, and V̇o2max after training were compared between athletes with initially normal ferritin and initially low ferritin (as defined above in Iron Stores). To assess the effects of a 4 wk altitude training camp with formal iron supplementation on multiple dependent variables, in Study II, serum ferritin concentration, Hb, RCV, and V̇o2max after training were compared between groups living at altitude and sea level. Differences in variables among groups at baseline were determined using one-way ANOVA. The effects of each intervention on variables were determined using one-way or two-way ANOVA with repeated measures. Bonferroni corrected t tests were used for multiple comparisons during post hoc testing. If the data failed the test for a normal distribution, a one-way ANOVA on ranks (Kruskal-Wallis) was applied. Post hoc comparisons for the nonparametric analyses utilized Dunn’s method. Spearman’s rank-order correlation coefficient was used to determine the relationship between changes in each two variables of Hb, RCV, and V̇o2max in Study II because these changes were not normally distributed while showing a monotonic relationship. A P value of < 0.05 was considered statistically significant. Data were analyzed using IBM SPSS version 6.1 (Armonk, NY).

RESULTS

Retrospective Study (Study I)

Before training camp, using our criterion levels for iron deficiency, 10 subjects (8 men and 2 women) had normal ferritin levels, whereas 9 subjects (5 men and 4 women) had low ferritin levels. As shown in Table 1, there were no differences in subject characteristics or dietary iron intake between athletes with normal and low ferritin levels. Thus, we observed a high prevalence of low iron stores within our cohort of competitive distance runners (38% of males and 67% of females), despite normal dietary iron intake.

Table 1. Characteristics of subjects in Study I and Study II just before training camp

n (men/women)Age, yrHeight, cmWeight, kgBody fat, %Dietary Elementary Iron Intake, mg/24 h
Study I
    Normal ferritin10 (8/2)22.1 ± 3.6174.9 ± 9.662.0 ± 7.06.2 ± 3.623 ± 9
    Low ferritin910 (5/4)22.2 ± 3.4174.9 ± 5.663.7 ± 7.49.4 ± 4.719 ± 4
Study II
    Altitude group2610 (18/8)21.9 ± 3.1174.8 ± 7.963.0 ± 6.78.2 ± 5.627 ± 11
    Sea-level group1310 (9/4)21.2 ± 1.3171.2 ± 9.760.1 ± 7.58.5 ± 5.018 ± 6

Values are means ± SD; n = number of subjects. Normal ferritin, serum ferritin level > 30 ng/mL for men and > 20 ng/mL for women. Low ferritin, serum ferritin level ≤ 30 ng/mL for men and ≤ 20 ng/mL for women.

Serum ferritin, Hb, RCV, and V̇o2max before and after altitude training camp are presented in Table 2. Before the training camp, athletes with low ferritin levels had lower Hb than athletes with normal ferritin levels, although there were no differences in RCV or V̇o2max. Interestingly, after the training camp at altitude, serum ferritin dropped, whereas Hb and RCV increased in athletes with normal ferritin levels and remained unchanged in athletes with low ferritin levels. Moreover, V̇o2max increased (P = 0.003) in athletes with normal ferritin levels and remained unchanged in athletes with low ferritin levels (P = 0.23).

Table 2. Effects of 4 wk of altitude training camp without formal iron supplementation in athletes with initially normal or low ferritin levels

Normal Ferritin (n = 10)
Low Ferritin (n = 9)
Pre-AltitudePost-AltitudePre-AltitudePost-Altitude
Serum ferritin, ng/mL69 ± 3034 ± 18*18 ± 10#12 ± 8#
[Hemoglobin], g/L142 ± 1148 ± 1*135 ± 1#139 ± 1#
Red cell mass, mL/kg27.3 ± 3.129.8 ± 2.4*27.0 ± 2.927.5 ± 3.8
Maximal oxygen uptake, mL⋅kg−1⋅min−162.6 ± 3.166.2 ± 3.7*60.0 ± 7.362.2 ± 7.5

Values are means ± SD; n = number of subjects. Normal ferritin, serum ferritin level > 30 ng/mL for men and > 20 ng/mL for women. Low ferritin, serum ferritin level ≤ 30 ng/mL for men and ≤ 20 ng/mL for women.

*P < 0.05 comparing pre-altitude vs. post-altitude within group;

#P < 0.05 compared with athletes with normal ferritin levels.

All measurements were made at sea level.

Based on Eq. 2, the change in ferritin concentration over 4 wk at altitude was 22.5 ng/mL (0.8 ng·mL−1·day−1). Thus, daily iron used at altitude was 6.4 mg/day. Based on Eq. 1, the additional daily iron requirement for altitude training was 4.9 mg/day (1.9 mg/day required for training + 6.4 mg/day used at altitude – 3.4 mg/day dietary intake).

Prospective Study (Study II)

Fourteen subjects (8 men and 6 women) in the altitude group and 6 subjects (4 men and 2 women) in the sea-level group had low ferritin levels at study initiation, before the start of formal iron supplementation. All of the athletes had normal ferritin levels before the start of field training camp, and none showed low ferritin levels during the camp.

As shown in Table 3, serum ferritin remained unchanged in response to the field training camp at altitude supported by high-dose liquid iron supplementation, suggesting that this strategy of ferritin-guided iron replacement was adequate to provide sufficient iron stores required for athletes training at altitude. In contrast, serum ferritin in the sea-level group increased during the field training camp. Hb in the altitude group increased during the field training camp and remained elevated after descent. Conversely, Hb in the sea-level group remained unchanged after return to Dallas, although it showed a minor increase during field training camp. RCV and V̇o2max increased after descent in the altitude group but were unchanged in the sea-level group. Most importantly, RCV and V̇o2max, after descent were higher by ~11% and 5.5%, respectively, in the altitude group compared with the sea-level group.

Table 3. Effects of 4 wk of altitude training camp with formal iron supplementation hematological parameters and maximal oxygen uptake

Sea-Level TrainingField Training Camp
Before Sea-Level TrainingAfter 4 wkAfter 36 hAfter 2 wkAfter 4 wkAfter Descent
Altitude group, n = 26
    Serum ferritin, ng/mL28 ± 1728 ± 1525 ± 1129 ± 1434 ± 1830 ± 18
    [Hemoglobin], g/L135 ± 8136 ± 8146 ± 8*151 ± 9*148 ± 8*
    Red cell mass, mL/kg27.1 ± 3.528.4 ± 3.830.6 ± 4.3*
    Maximal oxygen uptake, mL⋅kg−1⋅min−163.2 ± 5.365.9 ± 5.167.3 ± 5.6*
Sea-level group, n = 13
    Serum ferritin, ng/mL37 ± 1729 ± 1525 ± 1447 ± 14*48 ± 13*43 ± 12*
    [Hemoglobin], g/L136 ± 10139 ± 9147 ± 8*147 ± 11*140 ± 11
    Red cell mass, mL/kg28.2 ± 6.627.9 ± 3.427.6 ± 3.8
    Maximal oxygen uptake, mL⋅kg−1⋅min−164.3 ± 6.564.6 ± 6.763.8 ± 6.5

Values are means ± SD; n = number of subjects.

*P < 0.05 compared with after 4 wk of sea-level training within group.

P < 0.05 compared with sea-level group.

Spearman’s rank-order correlation matrix between changes in each variable is shown in Table 4. There was a strong and statistically significant relationship between RCV and Hb [r = 0.525, 95% confidence interval (CI): 0.251–0.721, P < 0.005] and between RCV and V̇o2max (r = 0.368, 95% CI: 0.059–0.612, P = 0.022).

Table 4. Spearman’s rank-order correlation matrix between changes in each variable from pre- to post-altitude

ΔRed Cell MassΔ[Hemoglobin]
Δ[Hemoglobin]
    r0.525
    P<0.005
    n39
ΔMaximal oxygen uptake
    r0.3680.332
    P0.0220.039
    n3939

Data on the relationships between changes in each variable from before to after the field training camp are presented.

DISCUSSION

The major findings in this study are as follows: 1) RCV and V̇o2max increased in response to 4 wk of altitude exposure in runners with initially normal iron stores but did not increase in runners with initially low iron stores without formal iron supplementation, and 2) 4 wk of altitude exposure after treatment of low iron stores with high-dose liquid iron supplementation and confirmation of adequate iron availability increased RCV, which resulted in a greater increase in V̇o2max. Thus, low iron stores before an altitude sojourn prevent erythropoiesis and consequently an improvement in sea-level athletic performance; this deficit can successfully be treated by high-dose liquid iron supplementation preceding and during altitude exposure.

Iron Availability and Responsiveness to Altitude Exposure

Govus et al. (32) retrospectively analyzed data from 178 athletes exposed to low to moderate altitude (1,350–3,000 m) for 2–4 wk. They found that serum ferritin levels decreased in athletes supplemented with nil or 105 mg oral iron daily, serum ferritin increased in athletes supplemented with 210 mg oral iron daily during altitude exposure, and the change in Hb mass after altitude was higher in athletes who supplemented with higher doses of oral iron. With these data, the authors suggested that oral iron supplementation during altitude exposure may enhance Hb mass production and assist the maintenance of iron balance in athletes with initially low iron stores. The present study complements and extends these findings by clearly demonstrating in both the retrospective and prospective studies that adequate iron availability, which was attained by oral iron replacement titrated by serum ferritin levels, was required for effective erythropoiesis to hypoxic exposure during altitude training in competitive runners. Govus et al. (32) also demonstrated the importance of iron supplementation during an altitude training camp; in addition to this work, in our Study II, we demonstrated the importance of normalizing iron stores before commencing an altitude training camp. It is conceivable that having filled iron stores before altitude exposure is more important than supplementation during altitude exposure. We judiciously speculate that this may be because the ferrokinetics are probably quicker if iron is taken up by the erythroblasts from the available iron in macrophages with normalized iron stores before altitude exposure versus iron having to first traverse the intestine, as is the case with starting oral iron supplementation after the sojourn to altitude has begun (37).

It is well established that iron is an indispensable component of hemoglobin synthesis, and low iron stores prevent effective erythropoiesis and consequently result in physiologically relevant iron deficiency, with or without anemia at sea level in athletes as well as nonathletes (4, 46, 64). During altitude sojourns, iron requirements and consumption are enhanced due to the acceleration of erythropoiesis to hypoxia, which has been demonstrated previously by a decrease in serum ferritin concentration (36, 61), an increase in total iron-binding capacity (36), a decrease in transferrin saturation, and an increase in iron absorption in the digestive system (58) during altitude training in athletes. We also observed in the retrospective study a marked fall in serum ferritin concentration in athletes, even in those with initially normal ferritin levels, presumably from increased iron utilization. In contrast, athletes with initially low ferritin levels had minimal further reduction at altitude and did not increase Hb and RCV (Table 2). These findings support the notion that the failure to observe an increase in hematological parameters after a period of altitude training with an effective dose of altitude should raise the suspicion of low iron stores in such athletes. We speculate that the wide individual variation in the erythropoietic response to altitude training camps (19) can be accounted for, at least in part, by the variation of iron status of athletes at the beginning of and during altitude sojourn.

Hepcidin, a hepatic peptide hormone, plays the principal role in iron regulation and homeostasis (21, 53). As hepcidin is downregulated, it acts to enhance absorption of dietary iron by enterocytes and export of iron from reticuloendothelial macrophages into the plasma, thereby increasing plasma iron availability (21, 37, 53). Importantly, hepcidin is known to be lower in athletes with low serum ferritin levels at rest (57), whereas it is upregulated by chronic inflammation (21, 37, 53) and intense exercise (33) in response to the inflammatory-driven increases in IL-6 or high iron loading (12). As shown in Table 1, the amount of dietary elementary iron intake was not different between runners with normal and low ferritin levels in the retrospective study. From these observations, we can speculate that baseline levels of hepcidin might be upregulated by mechanisms associated with chronic inflammation (21, 37, 53, 56), or that exercise-induced increases in hepcidin might be upregulated despite a suppressed resting level in runners with low ferritin even at baseline. However, this hypothesis must be considered only speculative because we did not measure hepcidin in the present study.

On the other hand, the erythropoietic stimulus of altitude exposure suppresses hepcidin levels (29, 33). Indeed, Govus et al. (33) showed that resting hepcidin levels were suppressed after 2 and 14 days of normobaric hypoxia (3,000 m simulated altitude). However acutely, 3 h after exercise, increases in hepcidin were similar under either normoxia or hypoxia and were not altered after 14 days of chronic hypoxic exposure in well-trained distance runners. Importantly, these processes require sufficient iron levels (25). Coupled with our observations, adequate systemic iron availability, both before and during altitude, is a principal prerequisite to adapt erythropoietically to hypoxia and may have important implications for physiological and performance-based altitude training outcomes in endurance athletes (12).

Although contentious, it has been suggested that improved sea-level athletic performance after altitude training is associated mainly with improved oxygen transport to the working muscles during submaximal and maximal exercise (2, 40), secondary to an increased oxygen carrying capacity of blood with increased RCV and Hb (10, 43, 48, 62, 73, 74). Several previous studies have reported supportive evidence that V̇o2max or endurance performance at sea level does not significantly improve after altitude training when erythropoietic effect of an altitude was not observed (47, 65) or the accelerating erythropoiesis is prevented by phlebotomy (23). Indeed, the present data demonstrate that the failure to increase V̇o2max at sea level after altitude training was caused by a failure to increase RCV as a result of initially low iron stores in some athletes, and that most importantly, the sequela were improved by normalizing the iron deficiency before and during altitude sojourn (Table 3). From our observations in the prospective study, the predominant physiological effects of ‘altitude training’ of 2,500 m for 4 wk with adequate iron availability is an 11% increase in RCV, which significantly correlates to a 5% improvement in sea-level V̇o2max.

As for other possible mechanisms underlying the individual variation in the erythropoietic and performance-enhancing effects of altitude training, there is also a substantial variability in the EPO secretion to hypoxia between athletes (10). For example, in previous studies we showed that athletes who improved 5,000 m-run time more than 14.1 s after 4 wk of altitude training (termed responders) showed a significantly larger increase in EPO concentration from sea-level baseline after 30 h at altitude compared with athletes who did not improve 5,000 m-run time (nonresponders) after chronic altitude training. Additionally, after 14 days at altitude, EPO was still elevated over sea-level baseline in responders but was not significantly different from sea-level values in nonresponders. This EPO response led to a significant increase in RCV and V̇o2max in responders; in contrast, nonresponders did not show an increase in RCV or V̇o2max after altitude training. The wide individual variation of EPO response to hypoxia appears to be associated not only with the variation in O2 delivery to EPO producing tissues (26) and/or genetic backgrounds of athletes (38, 45, 66), but also with the variation in systemic inflammation, which directly and indirectly affects the EPO response to hypoxia at rest (18, 21, 56) and/or in response to exercise with hypoxic stimulation (56).

Recommendation for Athletes at Altitude

We observed a high prevalence of low iron stores within our cohort of competitive distance runners (38% of males and 67% of females), despite normal dietary iron intake, that is substantially higher than previous reports (11, 54). Low iron stores in athletes are associated with a number of factors, such as low iron intake and poor intestinal absorption, increases in total body Hb, myoglobin and cytochromes with exercise training, gastrointestinal bleeding (68), mechanical hemolysis by high blood flow and foot strike (50), and increased iron loss with excessive sweating (14). Daily iron requirements for typical endurance training, particularly running, are 2.3 mg for women and 1.8 mg for men at sea level (5). The additional daily iron requirement for erythropoiesis during altitude training calculated from the retrospective study was 4.9 mg/day. Therefore, calculated iron requirements for endurance athletes are increased from ~2 mg/day during training at sea level to 7 mg/day during training at altitude. In the prospective study, runners who had normal ferritin levels took elemental iron from 44 mg/day up to 264 mg/day and successfully maintained ferritin levels after training at altitude. In contrast, runners who had low ferritin levels required much higher doses of elemental iron, from 44 mg/day up to 396 mg/day, and slightly increased ferritin levels after training at altitude. Thus, our data suggest that high-dose liquid iron supplementation is effective for runners regardless of initial iron stores.

Moretti et al. (52) suggested that in healthy nonanemic young women with plasma ferritin ≤ 20 ng/mL, acute consecutive-day dosing results in decreased iron bioavailability (decreased relative fractional absorption) because of increased hepcidin levels and that moderate (40–80 mg) dose iron given on alternate days may maximize dosage efficacy and improve tolerance of iron supplements. Indeed, they showed that in healthy but iron-depleted (serum ferritin ≤ 25 ng/mL) young women, cumulative fractional and total iron absorptions with iron supplements (60 mg iron at morning) were higher in the alternate-day group than in the consecutive-day group (69). They further showed that twice-daily divided doses resulted in a higher serum hepcidin level than single-morning doses, although there were no significant differences in fractional or total iron absorptions between two dosing regimens (69). However, it should be noted that although the relative bioavailability of iron (i.e., fractional absorption) was decreased with higher doses of iron supplementation in these studies, the absolute amount of iron absorbed increased with increasing dose because of the larger total iron intake (52), suggesting that although our high-dose strategy likely increased hepcidin levels, absolute amounts of iron absorbed remained high. Moreover, these data are reported in nonathletes at sea level in an acute dosing study; however, we caution that any haphazard, high-dose iron supplementation protocol for athletes at altitude would not be appropriate and may pose a hazard. Measurement of serum ferritin levels every 2–4 wk during treatment will allow titration of iron dose and identify athletes heterozygous or homozygous for hemochromatosis (76). We recommend that doses be titrated to a target level of serum ferritin of 40–90 ng/mL. These calculations are specific for sea-level athletes spending 4 wk at moderate altitude. The iron requirements for fully acclimatized athletes living at these altitudes year round may be different, and requirements for such athletes should be determined by regular testing of serum ferritin.

Recently, intravenous iron replacement has become widely available for athletes with iron deficiency anemia as well as clinical patients as an alternative to oral replacement regimen (3, 12, 24, 55). However, intravenous iron preparation has a risk of anaphylactic shock and serum sickness reactions, although this risk is now exceedingly low with modern intravenous preparations (1). In the authors’ opinion, intravenous iron should be considered most strongly for those athletes with very low ferritin levels combined with manifest impaired performance and/or for those who cannot tolerate the doses of oral iron required to replace the very large iron deficits in such athletes. The dosage of intravenous iron for athletes with iron deficiency anemia should be titrated based on their Hb concentration and body weight, as is done for clinical patients (22, 24). Meanwhile, athletes with low ferritin levels without anemia should have dosage titrated based on their projected iron loss while at altitude and present ferritin level; 8 mg of iron should be added to increase 1 ng/mL of serum ferritin concentration because bioavailability of intravenous iron is 99% (33).

Conclusions

Exposure to moderate altitude places great demands on iron stores for erythropoiesis. Our data demonstrate that iron deficiency in athletes prevents accelerated erythropoiesis to 4 wk of moderate altitude exposure, mitigating improvement of V̇o2max and athletic performance on return to sea level. High-dose iron supplementation preceding and during altitude exposure can provide sufficient iron to allow effective erythropoiesis and improvement of V̇o2max and athletic performance at sea level. Thus, if an altitude sojourn is contemplated for competitive athletes, serum ferritin levels should be monitored and appropriate dose iron supplementation instituted to maximize the benefit for altitude exposure.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

J.S.-G. and B.D.L. conceived and designed research; J.S.-G. and B.D.L. performed experiments; K.O., J.S.-G., R.F.C., and B.D.L. analyzed data; K.O., J.S.-G., R.F.C., and B.D.L. interpreted results of experiments; K.O. prepared figures; K.O. drafted manuscript; K.O., J.S.-G., R.F.C., and B.D.L. edited and revised manuscript; K.O., J.S.-G., R.F.C., and B.D.L. approved final version of manuscript.

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AUTHOR NOTES

  • Address for reprint requests and other correspondence: B. D. Levine, Institute for Exercise and Environmental Medicine, 7232 Greenville Ave. Suite 435, Dallas, Texas 75231 (e-mail: ).