Effect of the Energy Intake on the Iron Status of Resistance Exercises Performed in Rats

In many cases, athletes compensate for nutrient deficiencies due to a reduced dietary intake by taking supplements or other means. However, in what ways nutrients are utilized by the body when it is deficient in energy and yet receives adequate amounts of the required nutrients are unclear. We therefore examined the effect of the balance between available energy and iron intake on the iron nutritional status of athletes. The experiment was conducted in two parts. Four-week-old male rats were divided into two groups based on energy and iron sufficiency: Experiment 1 was energy-sufficient and iron-sufficient (ES-FeS) and energy-sufficient and iron-deficient (ES-FeD). Experiment 2 was energy-deficient and iron-sufficient (ED-FeS) and energy-deficient and iron-deficient (ED-FeD) groups. All rats were made to perform climbing exercises 3 days a week at 5 P.M. The results showed that a significantly higher hematocrit, hemoglobin, plasma iron concentration, and TfS were found in the iron-sufficient group than in the iron-deficient group, TIBC was significantly lower in the iron-sufficient group than in the iron-deficient group, and TfS was significantly higher in the iron-sufficient group than in the iron-deficient group, irrespective of energy intake. It was suggested that restricting both iron and energy intake may significantly decrease the amount of iron in the liver and accelerate the metabolic turnover of red blood cells, while restricting iron intake but providing adequate energy intake suggested that resistance exercise–induced tissue iron repartitioning was not altered by iron sufficiency or deficiency.


Introduction
In general, it is believed that individuals performing intense exercise training have higher iron requirements than nonexercisers due to loss from heavy sweating (including shed epidermal cells), reduced absorption of iron from the gastrointestinal tract [1], and blood cell destruction [2] due to severe physical impact to the legs and other parts of the body, depending on the exercise category [3][4][5][6][7].However, female athletes often attempt to lose weight by reducing their dietary intake, as a thin body shape is often considered advantageous for performance and because they are concerned about esthetics [8].Therefore, iron deficiency anemia, which is most frequently observed among female athletes, has been suggested to be due to an inadequate iron intake caused by a reduced food intake [9,10].Although iron deficiency is most common in female athletes (approximately 15-35% of female athlete cohorts are deficient), approximately 5-11% of male athlete cohorts also present with this issue [11].Furthermore, Coates et al. reported that male triathletes and runners had a higher incidence of IDA than their female teammates, a finding that has not been previously reported [12].
The American College of Sports Medicine [13] and the International Olympic Committee [14] have pointed out that female athletes who continue to train intensely can have low energy availability due to their high energy expenditure from exercise, even in the absence of obvious eating disorders, and that continuous lack of available energy may risk amenorrhea and osteoporosis.
Athletes often use supplements to compensate for nutrient deficiencies from their reduced dietary intake.Indeed, approximately 50% of athletes report consuming some form of micronutrient supplement [15].However, how nutrients are utilized by the body when it is deficient in energy but receives adequate amounts of necessary nutrients is unclear.In addition, whether or not iron is excreted from the body without being absorbed or stored in the body is also unclear.
We therefore examined the effects of the balance between available energy and iron intake on the iron nutritional status of athletes.This study should serve as a reminder not only to athletes but also to women in general who have a low energy intake and supplement their diet with nutrients.

Experiment 1
The hypothesis that, if energy intake is adequate, deficient iron intake is associated with a reduced risk of anemia, will be considered.
The body weight of the rats was measured twice a week, and the food intake was measured everyday throughout the experiments.After the 21-day experimental period, the rats were fasted overnight and killed by drawing blood from the aorta abdominals at 8 A.M., under anesthesia with diethyl ether.The liver, spleen, kidneys, heart, flexor hallucis longus muscle (FHL), perirenal adipose tissue, posterior abdominal adipose tissue, mesenteric adipose tissue, and genital adipose tissue were quickly removed, weighed, and stored at − 30 °C until analyses.
The protocol of this study was approved by the Experimental Animal Care Committee of Osaka Aoyama University (No. 42).

Exercise
All rats performed a climbing exercise from 5 to 6 P.M., 3 days a week (once every 2 days) for 3 weeks, as previously reported [17][18][19].In brief, 10 rats were exercised simultaneously in a wire mesh cage (width 29 × depth 39 × height 100 cm).The bottomless cage was placed on an electric hot plate.The temperature of the hot plate was adjusted to 53 °C with an electrical transformer (SLIDAC SD 105; Toshiba, Tokyo, Japan).The rats moved around and crowded on the wall surface and were rarely positioned at the bottom of the climbing apparatus during the exercise.The temperature of 53 °C is the minimum temperature that can be used to provoke rats to climb without inducing burn injury in the event that the rats descend onto the hot plate.
The exercise regimen consisted of three sessions a week (5 min × 6 sets/day) during the 3-week study period.The rats were allowed to rest for 5 min between the training sessions.The exercise regimen was chosen after determining that the rats were able to continue exercising voluntarily without too much fatigue and that the regimen induced neither significant changes in the adrenal weight nor decreases in the food intake or body weight gain due to intensive exercise in our preliminary study.In addition, the exercise regimen increased the mass of skeletal muscles, such as the flexor hallucis longus (FHL).

Analyses
The hematocrit levels were measured using blood samples collected into heparinized microcapillary tubes via centrifugation at 12,000 rpm for 5 min.The blood hemoglobin concentration was determined using the Fuji Drychem System (Fuji Drychem Slide HB-WII; Fujifilm, Tokyo, Japan).The plasma iron concentration was determined using the Nitroso-PSAP method (Metalloassay Iron Determination LS; Funakoshi, Osaka, Japan).The total iron binding capacity (TIBC) was measured according to the method recommended by the International Nutritional Anemia Consultative Group.The transferrin saturation (TfS) was calculated together with the plasma iron concentration and TIBC [20].

Tissue Iron Content
The liver, spleen, carcass, and skin were lyophilized, ground, and dissolved in 0.28 mol HCl.The solution was centrifuged, and the supernatant was subjected to the Nitoso-PSAP direct method to determine tissue iron content.

Statistical Analyses
The data are expressed as the mean ± standard deviation (SD).Statistics analyses were performed using Student's t test at 5% significance level of the difference.

Results
The body weight and food intake of the rats are shown in Table 1.There were no marked differences between ES-FeS and ES-FeD in the food intake or body weight at sacrifice during the 3-week period.The amount of iron obtained from the 3-week dietary intake was significantly higher in ES-FeS than in ES-FeD.
The values of hematocrit, hemoglobin, plasma iron, TIBC, and TfS are shown in Table 2.The hematocrit and hemoglobin levels were significantly higher in ES-FeS than in ED-FeD.
The TIBC was significantly lower in ES-FeS than in ES-FeD.The plasma iron and TfS did not differ significantly between the groups.
The wet weights of tissues are shown in Table 3.The gastrointestinal tract weight was significantly lower in ES-FeS than in ES-FeD.There were no significant differences in the liver, spleen, kidney, adrenal gland, heart, gastrocnemius, plantaris muscle, or FHL weights.The retroperitoneal and intra-abdominal fat weights were significantly higher in ES-FeS than in ES-FeD.The perirenal fat, genital fat, and mesenteric fat weights tended to be higher in ES-FeS than in ES-FeD.
The dried tissue iron content is shown in Table 4.The skin content was significantly lower in ES-FeS than in ES-FeD.The carcass was significantly higher in ED-FeS than in ED-FeD.The iron content in the liver, spleen, and muscle was not significantly different between the groups.The spleen iron content tended to be higher in ES-FeS than in ED-FeD.

Experiment 2
Examining the effects of restricted energy intake on the iron nutritional status of rats performing resistance exercise.
The other breeding method and exercise procedures, analysis of the blood and plasma components, diet composition, measurement of tissue iron content, and statistical processing were the same as in experiment 1.

Results
The body weight and food intake of the rats are shown in Table 5.There were no marked differences between ED-FeS and ED-FeD in the dietary intake or body weight at sacrifice during the 3-week period.The amount of iron obtained via the diet for 3 weeks was significantly higher in ED-FeS than in ED-FeD.
The hematocrit level, blood hemoglobin concentration, plasma iron, TIBC, and transferrin saturation (TfR) are shown in Table 6.The hematocrit level, hemoglobin concentration, plasma iron, and TfS values were significantly higher in ED-FeS than in ED-FeD; the TIBC did not differ significantly between the groups; and the TfR was significantly higher in ED-FeS than in ED-FeD, with no significant difference in the TIBC between the groups.
The wet weights of tissues are shown in Table 7.The digestive tract weight was significantly lower in ED-FeS than in ED-FeD.The soleus muscle weight was significantly higher in ED-FeS than in ED-FeD.However, the total skeletal muscle mass did not differ markedly between the groups.The mesenteric and abdominal fat weights were significantly lower in ED-FeS than in ED-FeD.There were no significant differences between the groups in the liver, spleen, kidney, adrenal, heart, gastrocnemius, plantaris, FHL, perirenal fat, peri-genital fat, or posterior abdominal wall fat weight.
The dried tissue iron content is shown in Table 8.The iron content in the carcass and liver was significantly higher in ED-FeS than in ED-FeD.The skin and spleen, FHL iron content tended to be lower in ED-FeS than in ED-FeD.

Discussion
It is conceivable that iron intake as well as energy sufficiency is necessary to improve iron deficiency anemia.Therefore, the effects of the balance between available energy and iron intake on the iron nutritional status of rats performing resistance exercise were examined in experiments 1 and 2. In experiment 1, whether or not a low iron intake can prevent anemia if energy intake is sufficient was examined.The results showed that the hematocrit and hemoglobin values were significantly higher for the FeS than for the FeD, which suggests that anemia cannot be prevented if the iron intake is low, even if energy is sufficient.This experiment did not measure the exercise capacity or endurance.Therefore, it is not possible to determine whether the reduction in exercise capacity due to iron deficiency was compensated for by energy sufficiency.It may be necessary to investigate whether or not iron deficiency reduces the decrease in athletic performance in the future.
Regarding the tissue weight, the gastrointestinal weight was significantly higher under conditions of iron deficiency than under conditions of iron sufficiency in both experiments 1 and 2. Iron is absorbed via the duodenum.It has also been suggested that resistance exercise has no effect on iron absorption [19].Therefore, it is possible that gastrointestinal enlargement enhances iron absorption.
There was significantly less intra-abdominal fat in ES-FeD than in ES-FeS.However, there were no significant differences in food intake between the groups in this study.A previous study reported that the absolute weight of intra-abdominal fat was reduced with an iron-deficient diet intake compared to a control diet intake [5,21].It has also been reported that the overall intake decreases when iron-deficient diets are consumed [22].The suppression of dietary energy intake results in an increase in circulating glucose levels, which in turn results in a reduced food intake.Because more glucose breakdown occurs under these conditions, iron-deficient animals are reported to be relatively energy-deficient as well [23].The decrease in intra-abdominal fat in cases of iron deficiency in this study was presumably due to abnormal energy metabolism.
The liver and spleen are deficient in stored iron at the stage where hemoglobin concentrations are reduced due to deficiency.However, in the present experiment, no differences were found between groups in the liver and spleen, nor in the muscles.We previously reported that resistance exercise alters iron redistribution.It has also been reported that iron is redistributed to tissues with increased oxygen consumption.In our study, both groups were allowed to exercise.The fact that no marked difference in tissue iron content was noted suggests that iron redistribution by resistance exercise may be similar, regardless of the amount of iron ingested.
Experiment 2 examined the effects of different energy intake restrictions on the iron status of rats performing resistance exercise.The hematocrit and hemoglobin levels, plasma iron, TIBC, and TfS blood indices all showed significant differences.This may be due to the low iron content in the diet.
The soleus and gastrocnemius muscle weights tended to be higher in ED-FeS than in ED-FeD.In contrast, the mesenteric and intra-abdominal fat weights were significantly higher in ED-FeS than in ED-FeD.In the previous experiment, it was inferred that iron-deficient animals would have decreased intra-abdominal fat due to abnormal energy metabolism.However, an inadequate energy intake in addition to iron deficiency increased the amount of intraabdominal fat and decreased muscle hypertrophy.Since there was sufficient iron in ED-FeS, it is thought that energy metabolism worked normally, and lipids were used as an energy source.In contrast, ED-FeD was deficient in both energy and iron and, as mentioned above, is considered to have abnormal energy metabolism.However, the consumption of lipids by energy metabolism exceeded the abnormal energy metabolism due to iron deficiency, which likely led to the lipid weight being lower in ED-FeD than in ED-FeS.
The tissue iron content was about twice as higher in ED-FeD as in ED-FeS, although there was no significant difference in the iron content in FHL.It has been reported that the distribution of iron to tissues increases with increased oxygen consumption due to exercise [24][25][26], and it is possible that the resistance exercise load was higher in ED-FeD than in ED-FeS.On comparing the tissue iron content, the amount of iron in the whole spleen tended to be higher in ED-FeD than in ED-FeS (p = 0.08).Old erythrocytes are broken down and reused in the spleen [27,28].Strenuous exercise reportedly causes weakening of erythrocyte membranes [14].The metabolic turnover of erythrocyte iron is said to be higher in animals that exercise under low-iron conditions than in those that do not exercise [21,22].These findings suggest that energy deprivation and iron deprivation may have caused erythrocyte membrane fragilization and accelerated erythrocyte metabolic turnover.

Conclusion
It was suggested that restricting both iron and energy intake may significantly decrease the amount of iron in the liver and accelerate the metabolic turnover of red blood cells, while restricting iron intake but providing adequate energy intake suggested that resistance exercise-induced tissue iron partitioning was not altered by iron sufficiency or deficiency.However, the effects of differences in energy intake on the iron nutritional status of resistance exercise rats could not be fully elucidated.Further investigations will be needed in the future.

Table 1
Final body weight, total food intake and total iron intake during the study

Table 2
Blood iron status parametersValues are the mean ± SD * p < 0.05, significantly different from ES-FeS (Student's t test)

Table 3
Tissue weight (g/wet mass of tissue) * p < 0.05, significantly different from ES-FeS (Student's t test)

Table 4
The iron content in various tissues (dry tissue) * p < 0.05, significantly differently different from ES-FeS (Student's t test)

Table 5
Final body weight, total food intake and total iron intake during the study

Table 6
Blood iron status parameters

Table 7 Tissue
weight (g/wet mass of tissue) Values are the mean ± SD FHL flexor hallucis longus * p < 0.05, significantly different from ED-FeS (Student's t test)

Table 8
The iron content in various tissues (dry tissue) Values are the mean ± SD FHL flexor hallucis longus * p < 0.05, significantly different from ED-FeS (Student's t test)