effects of a fixed dietary intake on changes in red blood ... · delta-aminolevulinate dehydratase...
TRANSCRIPT
597
International Journal of Sport Nutrition and Exercise Metabolism, 2006, 16, 597-610© 2006 Human Kinetics, Inc.
Sakuko Ishizaki and Jun Yamakawa are with Japan Womenʼs College of Physical Education, Tokyo 157-8565, Japan. Takako Koshimizu is with Japan Institute of Sports Sciences, Nishigaoka, Tokyo 115-0056, Japan. Kae Yanagisawa is with the Department of Human Nutrition, Faculty of Humanities, Seitoku University, Matsudo-city, Tiba Prefecture 271-8555, Japan. Yoshiko Akiyama, Yuko Mekada, Nobuyoshi Shiozawa, and Yukari Kawano are with the Department of Nutritional Science, Tokyo University of Agriculture, Tokyo 156-8502, Japan. Noriko Takahashi is with the Department of Health Care Science, Showagakuin Junior College, Ichikawa-city, Chiba 272-0823, Japan.
Effects of a Fixed Dietary Intakeon Changes in Red Blood Cell
Delta-Aminolevulinate DehydrataseActivity and Hemolysis
Sakuko Ishizaki, Takako Koshimizu, Kae Yanagisawa,Yoshiko Akiyama, Yuko Mekada, Nobuyoshi Shiozawa,Noriko Takahashi, Jun Yamakawa, and Yukari Kawano
This study was to assess the effect of a fi xed dietary intake on biomarkers of red blood cell (RBC) biosynthesis and degradation. Over a two-year period, eight collegiate rhythmic gymnasts participated in this study. During the fi rst year, they ate self-selected diets. During the second year, a fi xed dietary intake involving consumption of common Japanese foods containing 15 mg iron and 1500 kcal energy was maintained for 4 wk at the beginning of the program. Fixed dietary intakes resulted in signifi cantly increased intakes of protein, minerals and vitamins, and signifi cantly decreased fat intake, but total energy and carbohydrate intakes were unchanged. Mean values of RBC, Hb, Ht, or TIBC were not affected by the intervention. A fi xed dietary intervention appeared to enhance RBC turnover by increasing the capacity for erythrocyte biosynthesis and degradation, although the prevalence of iron-defi ciency anemia remained unchanged.
Key Words: period of weight loss, iron defi ciency anemia, haptoglobin, erythro-cyte turnover
Competition in rhythmic gymnastics includes fi ve hand implements recognized by the International Gymnastics Federation: ball, hoop, clubs, ribbon, and rope. The athletes must perform physically demanding routines of 1 to 1.5 min duration with each of the fi ve implements. For rhythmic gymnasts to maximize performance, they strive to achieve an optimum sport-specifi c body size, low body mass and low body fat level (7, 11, 12, 17, 25). Leanness is particularly valued because the gymnastʼs success is a function of appearance as well as technical performance.
598 Ishizaki et al
The strategies rhythmic gymnasts use to achieve optimum weight and body composition may, however, predisposes them to a variety of medical complica-tions that include menstrual dysfunction, eating disorders, growth retardation, and nutritional defi ciencies such as iron-defi ciency anemia (4, 5, 11, 17, 25).
Iron defi ciency and anemia are recognized as the most frequently developed nutritionally preventable health problems for female athletes, especially runners and gymnasts (5, 23). Iron depletion is related to quantities of food and inherent densities of iron in the diet, as well as a decision to exclude meat from a diet (2). Dietary supplementation with minerals such as iron and zinc could be needed (6). However, it is still unknown how common iron-defi ciency anemia is among Japa-nese collegiate female rhythmic gymnasts and whether dietary iron would be of value in this population. Furthermore, it is not well understood how the metabolism and degradation of red blood cells (RBC) in well-trained gymnasts correspond with sessions of training.
In this study, we examined the prevalence of iron-defi ciency anemia during two 8-week periods in female Japanese rhythmic gymnasts. In addition, we assessed what changes occur in the blood parameters of gymnasts over time when kept on the same energy and iron intakes but with higher intakes of protein and lower intakes of fat. Direct comparative measurements of key factors affecting the biosynthesis and degradation of RBC during training are not readily apparent. We then estimated delta-aminolevulinate dehydratase (δ-ALAD) activity (16) and haptoglobin (hp) concentration as the biosynthesis and degradation indices of RBC.
Materials and MethodsSubjects
Over a two-year period, eight collegiate rhythmic gymnasts participated in this study, which was approved by the Ethical Committee of the Japan Womenʼs Col-lege of Physical Education both in 2001 (No. 2001-1) and in 2002 (No. 2002-3). All subjects volunteered and were informed of all potential risks and procedures before giving their written informed consent to participate in this study. All had normal blood pressure and normal menstrual cycles. To create appropriate testing conditions, all participants were instructed to refrain from strenuous activity for 24 hours and were asked to fast overnight before the day of blood sampling.
Experimental Protocols
Each data observation took place at fi xed intervals, as shown in Figure 1. Subjects were assessed at baseline (Pre-1), 4 weeks post-baseline (Pre-2), and 8 weeks post-baseline (Pre-3) during the pre-intervention year. One year later, intervention was initiated with observations at baseline (Int-1) and 4 weeks post-baseline (Int-2). Four weeks following the end of the dietary intervention, a post-intervention assessment took place (Post-1). Meal intervention was carried out as follows: a dietitian created 15 menus providing approximately 15 mg iron and approximately 1500 kcal energy intake per day. The athletes consumed these diets for 30 days. All experimental diets consisted of customary Japanese foods.
Effects of a Fixed Dietary Intake 599
All data regarding the blood samples, body weight, and dietary intakes were collected at each observation period from 1 to 2 weeks after the menstruation (Figure 1). On the morning of the day of blood sampling, subjects were asked to arrive at the laboratory between 7 and 8 AM following an overnight fast. On arrival, subjects completed the dietary assessments and the health claims. Height, body mass and body fat mass were measured (Model TBF 210, TANITA, Co., Tokyo, Japan) prior to the blood sampling.
Dietary Survey
Dietary data for continuous 3 days before the blood sampling day were obtained from each subject by a registered dietitian throughout the experimental period. The intakes of individual nutrients were calculated based on the Standard Tables of Food Composition for the Japanese (5th edition; 27).
Blood Analysis
Extreme care was taken during collection of blood to avoid the possibility of hemolysis; a 21-gauge needle was used together with minimal stasis. Blood was expelled down each of three separate collection tubes. Two milliliters of blood were collected in an EDTA-2K tube to assess hematological parameters. Five milliliters of blood were transferred into a serum tube and allowed to clot at room temperature. After centrifugation at 3,000 rpm, 4° C for 10 min, serum was col-lected. Blood for assessing plasma, osmotic fragility, and the activity of δ-ALAD were collected in a 2-ml heparin tube.
Figure 1—Experimental protocols. Over a two-year period, eight subjects completed all of the testing and training sessions. Data including body weight, blood samples, and dietary estimation were collected three times in Pre-1, Pre-2 and Pre-3 of the pre-intervention year, and in Int-1, Int-2 and Post-1 of the intervention year, respectively. A fi xed dietary intervention containing 15 mg iron and 1500 kcal energy was maintained for 4 wk during the periods of Int-1 to Int-2.
600 Ishizaki et al
Hematological parameters, including RBC counts, hemoglobin (Hb) concentra-tion, and hematocrit levels (Ht) were measured using an automated system (Horiba LC-360, Horiba Seisakusho KK, Japan). Concentrations of serum iron, ferritin, total iron binding capacity (TIBC), unsaturated iron binding capacity (UIBC), serum erythropoietin (EPO) and hp were measured at the Medical Lab Co. Ltd. (Tokyo, Japan) as follows: iron by atomic absorption spectrophotometry (SHIMAZU AA 6400-F, Shimazu Co. Ltd., Japan), ferritin and EPO by enzyme-linked fl uorescent assay, TIBC by the Nitroso - PSAP method, and hp by immune - nephelometry using a Nephelometry Analyzer II (Behring, Germany).
The osmotic fragility of RBC was run in duplicate and measured using the heparinized RBC fraction according to Beutler (3). δ-ALAD activity was run in duplicate, and assayed using the heparinized RBC fraction as described previously (13, 15, 16). Two ml of heparinized blood was centrifuged at 3,000 rpm for 10 min at 5 ºC and the resultant supernatant was discarded. The precipitated RBC were washed three times with 2 ml of cold phosphate buffered saline (pH 7.4), fi lled with the same buffer and stored at –80 ºC until assay. The resultant RBC was incubated at 37 ºC for 30 min with δ-aminolevulinic acid. The reaction was stopped with a TCA-HgCl
2 mixture and the color was developed using modifi ed Ehrlich reagent.
Enzyme activity was calculated using the molar absorption coeffi cient (6.1 x 104) of the fi nal Ehrlich color salt at 553 nm, and the results were expressed as porpho-bilinogen (nmol / mg Hb / hr).
Statistics
All values are presented as means ± standard deviations (SD). EXCEL version XP (Microsoft Corp., Japan) was used for data handling and graph generation. Data analysis by SPSS statistical software (version 11.5, SPSS Inc, JAPAN) was per-formed in three steps. Two-way (intervention x time) repeated measures analyses of variance (ANOVAs) were used to analyze dietary data, body iron status, hp, NaCL concentrations for osmotic fragility and δ-ALAD activity by means of Mauchlyʼs W test. Post hoc pairwise comparisons were by means of Friedmanʼs nonpara-metric ANOVA. After the fi nding of Friedmanʼs ANOVA was corroborated, the variables were submitted to a Wilcoxonʼs signed-rank test. Statistical signifi cance was accepted at P < 0.05.
Results
Physical Characteristics and Nutritional Intakes
Characteristics of the participants are shown in Table 1. The mean (± SD) age of the participants before the study was 18.6 (0.5) years. The mean weight, BMI and body fat percent did not change from Pre-1 to Pre-3 in the pre-intervention years, but those levels of the intervention year were signifi cantly lower in Int-2 than those in Int-1 (both, P < 0.05).
Both intakes of energy and carbohydrate were unchanged throughout the two-year experimental period, respectively (Table 1). In the pre-intervention year, intakes of most of the nutrients did not change over the three months. However,
Effects of a Fixed Dietary Intake 601
Tab
le 1
S
ub
ject
’s C
har
acte
rist
ics
and
Nu
trit
ion
al In
take
s
P
rein
terv
entio
n Ye
ar
Inte
rven
tion
Year
P
re-1
P
re-2
P
re-3
In
t-1
Int-
2 P
ost-
1
Cha
ract
eris
tics1
W
eigh
t (kg
) 51
.1 ±
5.1
51
.0 ±
5.5
51
.8 ±
5.4
51
.2 ±
3.7
a 48
.9 ±
2.7
b 50
.1 ±
3.2
b
B
MI
(kg/
m2 )
19
.7 ±
1.5
19
.7 ±
1.6
20
.0 ±
1.6
19
.9 ±
0.6
a 19
.1 ±
0.6
b 19
.6 ±
0.6
B
ody
Fat (
%)
21.2
± 3
.8
20.8
± 4
.2
20.7
± 3
.5
21.0
± 4
.0a
20.2
± 3
.5b
20.5
± 3
.3
Die
tary
inta
kes*
E
nerg
y (k
cal)
15
96 (
339)
15
62 (
394)
15
90 (
430)
15
55 (
330)
14
65 (
35)
1521
(39
8)
Fa
t (g)
6
0.2
(16.
4)
56
.2 (
19.4
)a
57.2
(22
.4)
65
.4 (
20.9
) a
27.6
(5.
7)b
54
.6 (
17.2
)a
Pr
otei
n (g
) 4
9.2
(12.
6)
49
.6 (
12.4
)a
50.8
(13
.4)
57
.9 (
15.2
)a
72.4
(6.
7)b
50
.8 (
20.8
)a
C
arbo
hydr
ate(
g)
212
(48)
2
11 (
48)
21
4 (4
5)
230
(90)
2
33 (
18)
200
(48
)
Ir
on (
mg)
7
.3 (
2.2)
6.4
(2.
5)a
6.
3 (1
.8)
6.1
(1.
5)a
1
8.2
(1.8
)b
6.7
(2.
6)a
V
itam
in C
(m
g)
84
(39)
77
(27)
78
(28)
73 (
34)a
21
2 (4
5)b
55
(30)
a
1 All
valu
es a
re e
xpre
ssed
as
mea
ns ±
SD
. N=
8. A
bbre
viat
ion:
BM
I; B
ody
Mas
s In
dex.
* All
valu
es a
re e
xpre
ssed
as
mea
ns (S
D).
Mea
ns w
ith th
e di
ffer
ent s
ubsc
ript
lette
s a
and
b ar
e si
gnifi
cant
ly d
iffe
rent
from
one
ano
ther
(P <
0.0
5) b
y W
ilcox
onʼs
sig
ned-
rank
test
.
602 Ishizaki et al
intakes of protein, calcium, magnesium, phosphate, iron, zinc, copper, vitamins A, E, K, B
1, B
2, B
6, B
12, and C were signifi cantly increased in Int-2, and intakes of fat
were signifi cantly decreased in Int-2 as compared with Pre-2, Int-1, and/or Post-1, respectively. The coeffi cient of variation in nutritional intakes throughout the study periods was smallest for energy (8.6%) and largest for Vitamin A (65.5%) in Int-2, respectively. The athletes were able to meet the energy (1500kcal) and iron (15 mg) targets during the intervention period.
Body Iron Status
Hematological Changes. There were no signifi cant differences observed in Hb, Ht, and TIBC levels among Pre-1, Pre-2, and Pre-3 (Table 2). MCV levels were signifi cantly higher in Pre-1 and Pre-2 than in Pre-3. MCH and MCHC levels were signifi cantly lower in Pre-2 and Pre-3 than in Pre-1. On the other hand, mean values of RBC, Ht, TIBC, and UIBC were signifi cantly lower in Int-2 than in Int-1, respectively. Mean values of MCV, MCH, and MCHC were lower in Post-1 than in Int-2, respectively.
Prevalence of Iron Depletion and Iron-Deficiency Anemia. There was no signifi -cant effect of this fi xed dietary intervention on the incidence of the iron-defi ciency anemia over the experimental period (Figure 2). Iron depletion, defi ned by a ferritin level below 20 μg/L, was found in fi ve, six, and three of the eight athletes in Pre-1, Pre-2, and Pre-3 of the pre-intervention year, and fi ve, four, and fi ve in Int-1, Int-2 and Post-1 of the intervention year, respectively. Iron-defi ciency anemia, defi ned by the presence of anemia (Hb levels below 12 g/dL), ferritin levels below 12 μg/L, and transferrin saturation ratio below 16%, was found in one or two athletes in every experimental period.
Serum iron concentrations and transferrin saturation ratios were unchanged throughout these experimental conditions (Figure 3). Serum ferritin concentra-tions were signifi cantly lower in Pre-2 than in Pre-1 (P < 0.05), but signifi cantly increased in Int-2 compared with those in Int-1, Post-1, and Pre-2 (both P < 0.05), respectively.
δ- ALAD Activity, EPO, hp and Osmotic Fragility. The activity of δ-ALAD was signifi cantly higher in Pre-3 than in Pre-1 (P < 0.05; Figure 4). The activity signifi cantly increased in Int-2 as compared to Int-1 (P < 0.001), Post-1 (P < 0.01), or Pre-2 (P < 0.05), respectively.
The EPO concentrations were signifi cantly higher in Pre-2 and Pre-3 than in Pre-1 (both P < 0.05), respectively, but those levels were unchanged among Int-1, Int-2 and Post-1. The EPO concentrations tended to be higher in the intervention year than in the pre-intervention year throughout the experimental periods.
There were no signifi cant differences observed in the serum hp concentrations between Pre-1, Pre-2 and Pre-3, respectively (Figure 5). The hp concentrations were the lowest in Int-2 and signifi cantly lower in Int-2 than those in Post-1(P < 0.05), but no signifi cant differences were observed between Pre-2 and Int-2.
The main effect of the intervention (P < 0.01), the main effect of the time (P < 0.01), and the intervention x time interaction effect (P < 0.05) on the osmotic fragility of RBC were all signifi cant. Mean NaCl concentrations as indices of
Effects of a Fixed Dietary Intake 603
Tab
le 2
H
emat
olo
gic
al C
han
ges
P
rein
terv
entio
n Ye
ar
Inte
rven
tion
Year
Vari
able
s P
re-1
P
re-2
P
re-3
In
t-1
Int-
2 P
ost-
1
RB
C (
x104 /
μl)
45
6 ±
26a,
1 47
4 ±
20b
480
± 26
b 49
1 ±
70 a
424
± 22
b#
461
± 33
Hb
(g /
dl)
12.9
± 0
.7
12.8
± 0
.9
12.9
± 1
.3
13.6
± 2
.1
11.9
± 1
.2#
12.2
± 1
.2
Ht (
%)
38.8
± 1
.8
39.9
± 2
.5
40.0
± 3
.2
41.5
± 6
.8a
35.0
± 2
.2b#
36
.9 ±
2.6
a
MC
V (μ
3 )
84.9
± 4
.2a
84.1
± 4
.5a
83.1
± 4
.9b
83.5
± 4
.3
82.2
± 5
.7a
80.2
± 6
.3b
MC
H (
rr)
28.5
± 1
.8a
27.0
± 1
.6b
26.9
± 2
.1b
27.6
± 2
.1
28.0
± 2
.5a
26.6
± 2
.8b
MC
HC
(%
) 33
.5 ±
1.1
a 32
.4 ±
0.5
b 32
.4 ±
0.9
b 33
.1 ±
1.3
34
.1 ±
1.4
a 33
.1 ±
1.4
b
TIB
C (
g/l)
31
4 ±
31 #
335
± 41
34
1 ±
48 #
418
± 38
a 39
4 ±
50 b
403
± 51
1 Not
e. V
alue
s ar
e m
eans
± s
tand
ard
devi
atio
n. N
= 8
.a,
b Mea
ns w
ith th
e di
ffer
ent s
ubsc
ript
lette
rs a
re s
ignifi c
antly
dif
fere
nt f
rom
one
ano
ther
with
in th
e sa
me
inte
rven
tion
peri
ods,
P <
0.0
5 (W
ilcox
onʼs
sig
ned-
rank
test
).# M
eans
of
the
sam
e tim
e w
ith o
r w
ithou
t int
erve
ntio
n ar
e si
gnifi
cant
ly d
iffe
rent
, P <
0.0
5 (W
ilcox
onʼs
sig
ned-
rank
test
).
604 Ishizaki et al
Figure 2—Comparison of the incidences of iron-defi ciency anemia. Body iron status was divided into three groups (Normal, Iron-defi ciency, Iron-defi ciency anemia) in Pre-1, Pre-2 and Pre-3 (Figure 2-1) of the pre-intervention year, and in Int-1, Int-2, and Post-1 of the intervention year (Figure 2-2), respectively. Abbreviations: Defi ciency; iron defi ciency, Anemia; iron defi ciency anemia.
osmotic fragility of RBC were 0.440±0.017%, 0.443±0.018%, and 0.429±0.023% in Pre-1, Pre-2, and Pre-3, respectively. NaCl concentrations were signifi cantly higher in Pre-2 than in Pre-3 (P < 0.05). On the other hand, each levels in Int-1, Int-2, and Post-1 were 0.481 ± 0.010%, 0.447 ± 0.028%, and 0.453 ± 0.036%, respectively. NaCl concentrations were signifi cantly lower in Int-2 than in Int-1 (P < 0.05), but not in Post-1.
DiscussionThis study of rhythmic gymnasts during a period of weight loss shows that diets containing 1500 kcal energy and 15 mg iron for one month might result in signifi -cantly lowered body weights and body fat percent (both P < 0.05) with increases in serum ferritin concentrations but not serum free iron and TIBC, and they do not
Effects of a Fixed Dietary Intake 605
Figure 3—Effect of the intervention on serum iron status. 1) Serum iron concentration (mean + SD), 2) Transferrin saturation ratio, and 3) Serum ferritin concentrations. No intervention x time interaction effect, no main effect of time, and no main effect of intervention were observed for either serum iron concentrations or transferrin saturation ratio (two-factor repeated-measures ANOVA). The main effects of the intervention (P < 0.05) and the time (P < 0.05) on serum ferritin concentrations were signifi cant, as was the intervention x time interaction effect (P < 0.01; two-factor repeated-measures ANOVA). Values marked “*” in Int-2 indicate signifi cantly higher concentrations than those in Pre-2 (Wilcoxonʼs signed-rank test; P < 0.01). a,b: Values with different superscript letters are signifi cantly different within the same year (Wilcoxonʼs signed-rank test; P < 0.05).
606 Ishizaki et al
Figure 4—Effect of the intervention on the activity of δ-ALAD and EPO concentra-tions. 1) Changes in the δ-ALAD activities (mean + SD) and 2) EPO concentrations. The effect of time (P < 0.001) and the intervention x time interaction effect (P < 0.001) were signifi cant in δ-ALAD activities (two-factor repeated-measures ANOVA). Values marked “*” in Int-1 or Int-2 indicate signifi cantly lower or higher concentrations than in the Pre-1 or Pre-2, respectively (Wilcoxonʼs signed-rank test; P < 0.05). a,b,c: Values with different superscript letters within the same year are signifi cantly different (Wilcoxonʼs signed-rank test; P < 0.05).
prevent the development of iron-defi ciency anemia. Furthermore, a fi xed dietary intake increased the δ-ALAD activity, but decreased the hp concentrations.
Rhythmic gymnastics is a competitive sport requiring speed, power, grace, and aesthetic appeal, so achievement of both a desired body shape and strength are necessary. Elite rhythmic gymnasts have been shown to be taller and leaner than average for their age (11, 12, 17). Rhythmic gymnasts sometimes consume less energy than the estimated requirements (6, 7).
Effects of a Fixed Dietary Intake 607
Under our experimental conditions, there were no signifi cant differences in total energy intakes over the experimental periods, with or without the intervention (around 1500 kcal/day). Thompson et al. (28) measured the resting metabolic rate in male endurance athletes who had either low energy intakes or adequate energy intakes. They found that the resting metabolic rates were signifi cantly lower in the low energy intake-group compared to those of the adequate intake-group, while physical activity levels of both groups were similar. These observations indicate that decreased resting metabolic rates are related to lower energy intakes.
In the pre-intervention year, female athletes took 1500 kcal/day of energy for the 8 weeks and both their body weight and body fat percent were unchanged throughout the period, suggesting that the energy intakes are suffi cient for the energy expenditures during the 8 weeks. On the other hand, athletes in Int-2 showed signifi cantly lowered body weight and body fat percent than in Int-1. Body weight and body fat usually decrease when energy expenditure exceeds energy intake. Therefore, it seems likely that energy expenditures in Int-2 are higher than those in Pre-2 and Int-1, respectively.
Furthermore, dietary iron intakes in Pre-1, Pre-2, Pre-3, Int-1, and Post-1 were approximately 6-7 mg/day, indicating that iron intakes were much lower than those reported in the National Survey of Nutrition in 2002 (19) or the Dietary Refer-ence Intakes for Japanese young women (20), respectively. Low energy intakes are associated with iron defi ciency in female athletes (2, 5, 24), while suffi cient iron intakes reduce iron-defi ciency anemia risk (29). Since dietary intakes of iron (14.6 - 15.0 mg) were reported to be suffi cient to prevent iron-defi cient anemia
Figure 5—Effect of the intervention on serum haptoglobin concentrations. Serum hp concentrations (mean + SD) in Pre-1, Pre-2 and Pre-3 of the pre- intervention year (white columns) or in Int-1, Int-2 and Post-1 of the intervention year (dark columns), respectively. The main effect of the time (P < 0.05) and the intervention x time interaction effect (P < 0.05) are signifi cant, but no main effect of the intervention was observed (two-factor repeated-measures ANOVA). a,b : Values with different superscript letters within the same year are signifi cantly different (Wilcoxonʼs signed-rank test; P < 0.05).
608 Ishizaki et al
(17), the minimum iron intake target in our present intervention program was established at 15 mg.
In the present study, iron depletion was found in 75% of the study population in Pre-1, 87.5% in Pre-2, and 62.5% in Pre-3, respectively. On the other hand, iron-defi cient anemia was found in only 14% of female basketball players (8). The incidence of iron depletion and iron-defi cient anemia in this study was much higher than in basketball players or endurance runners (8, 21), suggesting that rhythmic gymnasts may be more vulnerable to the iron-defi cient anemia than other athletes. However, it is not clear whether a 4-week dietary intervention is suffi cient to produce differences in the body iron profi les. Roughead et al. (22) examined the initial uptake and subsequent retention of heme and nonheme iron in human using isotopes, and found that at 2 weeks after the test meal intakes, ≈ 80% of the newly absorbed nonheme iron was incorporated into the erythrocytes of the subjects. This observation strongly suggests that the 4 week dietary intervention with Japanese ordinary foods in Int-2 provided enough substrate for increased erythrocytes bio-synthesis. However, in this period, enhanced physical activity might promote the destruction of erythrocytes more than the biosynthesis. Accordingly, the dietary intervention could not improve iron-defi cient anemia in Int-2.
Development of iron-defi cient anemia is attributed to some factors such as menses, lower dietary intakes of iron, foot strike hemolysis, blood and iron loss through the gastrointestinal tract and urinary tract, and by excessive sweating (8). However, it is not well understood how the metabolism and degradation of red blood cells (RBC) in well-trained rhythmic gymnasts correspond with sessions of training.
In our study, the activity of δ-ALAD was signifi cantly increased in Int-2 compared with Int-1 (P < 0.001) and Post-1 (P < 0.01), but hp concentrations were signifi cantly lower in Int-2 than in Post-1. The increase of red cell turnover in athletes was reported by Ehn et al. (10), in which whole-body loss of radioactive iron occurred ~20% faster in female athletes than in non-athletes. Increased activity of δ-ALAD in our present study may correspond with increased RBC turnover, because δ-ALAD, an enzyme in the prophyrin pathway, is the marker enzyme for heme biosynthesis.
The concentrations of serum EPO in Pre-2 and Pre-3, and the activity of δ-ALAD in Pre-3 increased signifi cantly compared with Pre-1, respectively. On the other hand, serum ferritin levels decreased signifi cantly in Pre-2. The decrease of serum ferritin may correspond to an increased biosyntheses of RBC in Pre-2. Although serum iron concentrations were unchanged, serum ferritin concentra-tions and δ-ALAD activities increased signifi cantly in Int-2 compared with both Int-1 and Post-1, respectively. There might be a possibility that serum iron is used as substrates for erythrocyte and ferritin biosyntheses and/or directly excreted in sweats and urine. The rise of serum ferritin concentration indicates that the iron of the meal origin has been absorbed.
Several studies have indicated that low hp levels are caused by the destruction of older RBC (18), and footstrike is the major contributor to hemolysis during running (26). Rhythmic gymnastic training involves extensive skipping and jumping with a club and/or a ball, and elite gymnasts would take advantage of a high anaerobic threshold in performing their competitive routines (1, 14). The vertical compo-nent of the grand reaction force had maximal amplitude of about 1900 N, equal to 3.5 times the force produced by the body weight of the gymnast (9). Under our
Effects of a Fixed Dietary Intake 609
experimental condition, lower hp was apparent in Int-2 of the dietary intervention periods. This suggests that the intense rhythmic gymnastic training might induce footstrike hemolysis just like runners. Furthermore, osmotic fragility indicated by NaCL concentrations was lower in Int-2, suggesting that this fi xed dietary intake might provide stronger and newer RBC against some changes in osmotic pressure and increased intensive activities.
In conclusion, the fi xed dietary intake did not improve iron depletion in female rhythmic gymnasts in this study. Some limitations in our present study include the fact that we only had 8 subjects. It is also unclear which of the factors directly affect the body iron status: namely, the periods of dietary intervention or the quantity of dietary iron intake. Although this fi xed dietary intake did not induce any changes in the incidence of iron depletion and/or iron defi ciency anemia, this fi xed dietary intervention reduces the RBC life span as indicated by an increased ALAD activity and a decreased hp concentration.
Acknowledgments
This work was supported by a Grant-in-Aid for Scientifi c Research C-2 (No. 13680163) from the Japan Society for the Promotion of Science (JSPS).
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