J. Nutr. Sci. Vitaminol., 29, 129-139, 1983

Characteristics of Gas in Response to Iron Treatment and Exercise in Iron-Deficient and Anemic Subjects

Y. OHIRA,1 D. R. SIMPSON,1 V. R. EDGERTON,1 G. W. GARDNER,1 and B. SENEWIRATNE2

1 Department of Kinesiology, University of California, Los Angeles, California, 90024 USA 2Department of Medicine , University of Sri Lanka, Peradeniya, Sri Lanka (Received June 14, 1982)

Summary The effects of iron deficiency anemia and iron treatment on blood gas and acid-base balance at rest and during exercise were studied. Eight Sri Lankan males and 13 females were randomly divided into an iron treatment or placebo group. Their initial (Hb) levels were 6.2•}0.5 and 5.5•}0.7g/100ml (mean•}SEM) with iron levels of 41•}8 and 30•}6ƒÊg/100ml, respectively. Hemoglobin concentration was increased significantly within 7 days after iron treatment to 7.2•}0.4g/ 100ml. Resting lactate was higher than normal, while base excess, buffer base, and actual were lower, maintaining a normal pH. Heart rates during exercise at a given work load and lactate production following exercise decreased after the elevation of Hb. Venous blood PCO2 rose as Hb concentration increased, preceeding a significant in crease in resting O2 content, 16 days after iron treatment. With anemia, O2 delivery is potentially maintained by a shift of the O2-dissociation curve to the right due, in part, to 2,3-diphosphoglycerate. There was no significant change in Po2 or Hb-O2% saturation following exercise or iron treat ment. These data suggest that severe iron deficiency anemia results in lactate accumulation in blood even at rest but pH is maintained within normal limits. It was also suggested that severe anemia may impair CO2 transport capacity of blood which could limit continuation of muscle . Key Words iron deficiency anemia, work performance, O2 and CO2 transport, acid-base balance

It has been shown that the greater the severity of anemia in humans and rats, the greater the decrement in work capacity (1-8). Decreased O2 carrying capacity of blood (7) and decreased O2 utilizing capacity of tissues (1, 3, 9-11, 26, 27) cause a 1 大 平 充 宣

129 130 Y. OHIRA et al.

decreased maximal capacity of O2 consumption (7). However, besides O2-related factors, lactic might also be a possible limiting factor for work performance in severe iron deficiency anemia (6, 12, 13). King and Mazal's data (14), that showed a lower PCO2 in blood with a lower hematocrit, suggest a decreased venous blood PCO2 in anemia. In previous studies (5,15), it was found that the subjects' most frequent reason for terminating a maximal work capacity task on a treadmill was because of discomfort in leg muscles, and infrequently due to breathlessness or attainment of maximal heart rate. These observations suggested that the limitations to work capacity in iron deficiency anemia may not be restricted to the O2 carrying capacity of the blood. Consequently, we conducted systematic study to investigate the characteristics of blood gas and acid-base balance both at rest and during exercise in iron-deficient and anemic subjects before and after iron treatment.

METHODS

In a pre- and post-iron treatment study, 8 Sri Lankan men (ages from 24 to 76) and 13 women (ages from 18 to 62) were studied (Table 1). All experimental procedures were conducted according to the guidelines adopted for patient care by the Faculty of the Kandy General Hospital. The experimental design was explained by a native doctor and voluntary consents were obtained. Subjects were allowed to withdraw from the study at any time. Whether they withdrew or not, all of the subjects were treated for iron deficiency anemia at the end of the study. The initial levels of Hb (cyanmethemoglobin method), (16), and serum total (refractometer) were checked on two separate days with a three-day interval. They were randomly divided into an iron treatment group (n=11) and a placebo group (n=10). Selected parameters were measured before and 2, 7, and 16 days after a single i.v. dose (30-50ml based on their hemoglobin, Hb, levels) of iron dextran (Imferon, Fisons Ltd., Holmes Chapel, England) or a saline placebo. A standard treadmill exercise was performed with increasing work load increments as described in our previous study (15). But unlike that study, the maximum work load attained by each subject prior to iron treatment was used as the end-point for each of the subject's subsequent post-treatment tests in order to standardize pre- and post-treatment comparisons. A transthoracic electrocardio

Table 1. Characteristics of the subjects.

Mean•}SEM.

J. Nutr. Sci. Vitaminol. BLOOD GAS IN IRON DEFICIENCY ANEMIA 131

gram (V5-RV6) was monitored throughout the test. Approximately 5ml of blood was withdrawn from the brachial vein after at least a 30-min seated rest and immediately after exercise. The following parameters were measured: Hb, 2,3-diphosphoglycerate (2,3-DPG, 17), lactate (18), pH, PO2, PCO2 and Hb-02% saturation (Instrumentation Laboratory blood gas and pH analyzer, Model 208-01, 213, 329, and CO-Oximeter, Model 182, Boston, Mass.). Partial pressure of O2 at 50% saturation of Hb (P50) was estimated at 37•Ž with a PCO2 of 40mmHg and pH 7.40, using samples taken before and after exercise. Base excess, buffer base, actual bicarbonate, and O2 content were also calculated.

RESULTS

Major causes of anemia in Sri Lanka are malnutrition or hookworm in festation as was reported by Senewiratne et al. (19). The subjects in the current study were also iron-deficient with serum iron levels of 41•}8 and 30•}6ƒÊg/100ml in the iron treatment and placebo group, respectively. The initial mean (•}SEM) Hb values were 6.2•}0.5 and 5.5•}0.7g/100ml for these groups (Table 2). Their serum protein levels were normal (7.7•}0.2g/100ml). According to the pre-test survey, their Hb levels prior to the initiation of the study were stable at least for a couple of weeks. The constant Hb levels in the placebo group up to the 16th day also support this.

The time course of Hb concentration following i.v. infusion of iron dextran or a saline placebo is shown in Table 2. The Hb content was improved significantly

(p<0.05) within 7 days after iron injection. No improvement was observed in the placebo group. A significant hemoconcentration following the standardized exercise was found in the iron treatment group before iron and in both groups 2 days after

treatment. Neither PO2 (Table 2) nor Hb-O2% saturation (Table 3) changed significantly

even though both at rest tended to increase following iron treatment and tended to decrease after the standardized exercise bout (p>0.05). Following the elevation of

Hb, the resting O2 content was significantly increased 16 days after iron injection

(Table 2, p<0.05). The PCO2 levels both before and after exercise were elevated when Hb levels were improved at the 7th and 16th day after iron treatment

(p<0.05), but it did not change significantly in response to exercise (Table 2). The correlation coefficient between PCO2 and Hb concentration was 0.44 (p<0.01).

The base excess, buffer base, and actual bicarbonate tended to be lower than normal even at rest in most of the cases (Table 4). And they tended to decrease

further following exercise. Statistically significant decreases were observed in many situations in base excess and buffer base (See Table 4). For an unknown reason the

pre-exercise base excess and buffer base in the placebo group significantly decreased on day 2 (p<0.01). All of these values tended to be elevated 16 days after iron

treatment even though statistical significance was found only in post-exercise bicarbonate (p<0.05). Furthermore, these parameters on both pre- and post

Vol. 29, No. 2, 1983 132 Y. OHIRA et al. Table 2. Changes in Hb PO2, O2 content and PCO2 in response to iron treatment and to a standard exercise .

Mean•}SEM. Numbers 0, 2, 7, and 16 in the first column mean the days after each treatment . T and P represent iron treatment and

placebo group, respectively. The •õisp<0.05 by Paired t-test between day 0 and each day after treatment. The *and **arep<0.05 and

P<0.01 respectively, by paired t-test between pre- and post-exercise. BLOOD GAS IN IRON DEFICIENCY ANEMIA 133 Table 3. Changes in 2,3-DPG, P50, and Hb-O2% saturation in response to iron treatment and exercise.

Mean•}SEM. Numbers 0, 2, 7, and 16 in the first column are the days after treatment. T and P mean iron treatment and placebo

group, respectively. The •õ isp<0.05 by Paired t-test between the values before and each day after treatment. The * shows p<0.05 b y paired t-test between pre- and post-exercise values. 134 Y. OHIRA et al.

Table 4. Changes in base excess, buffer base, and actual bicarbonate levels following iron treatment and exercise.

Mean•}SEM. Numbers 0, 2, 7, and 16 in the first column show the days after treatment. T and P represent iron treatment and placebo group , respectively. •õp<0.05 and •õ•õp< 0.01 by paired t-test between day 0 and each day after treatment. *p<0.05 by paired

t-test between pre- and post-exercise. ap<0.05, bp<0.01, and cp<0.001 by unpaired

t-test between T and P groups.

exercise 16 days after iron treatment were significantly higher than in the placebo

group. The 2,3-DPG levels were significantly changed at the 16th day following iron therapy (Table 3). The concentration expressed inƒÊmol/ml at the 16th day was

greater than pre-treatment level both before and after exercise (p<0.05). On the other hand, the pre-exercise level per gram Hb was significantly reduced (p<0 .05), The 2,3-DPG levels expressed in both ways tended to increase following the

standardized exercise, but statistical significance was observed only in the pre treatment placebo group (p<0.05). However, P50 values did not change signi fi cantly (Table 3).

Since the work load and total exercise time were constant in each subject for all tests, parameters were compared at the same work load. Heart rates during exercise

at a given work load gradually decreased following iron treatment (Table 5). A significant decrease was observed on the 7th (at moderate work load) and 16th days

(at low, moderate, and high work load). No change was found in resting heart rates

after iron treatment. Following the increased Hb (Table 2) and lowered heart rates , significant lactate elevation after exercise disappeared 7 and 16 days after iron treatment (Table 5). Post-exercise lactate levels in the placebo group were con

sistently higher than pre-exercise values. Resting blood lactate was elevated above

pre-treatment levels on the 2nd and 7th days after iron treatment. The resting venous blood pH was within normal range although the mean values were

J. Nutr. Sci. Vitaminol. BLOOD GAS IN IRON DEFICIENCY ANEMIA 135 Table 5. Responses of heart rates, venous blood lactate, and pH to iron treatment and exercise.

Mean•}SEM. Numbers 0, 2, 7, and 16 in the first column mean the days after each treatment. T and P represent iron treatment and

placebo group, respectively. The•õ, •õ•õ, and •õ•õ•õ are p<0.05, p<0.01 and p<0.001 by paired t-test between before and each day after treatment. The * p<0.05 and ** p<0.01 are the results of paired t-test between pre- and post-exercise. Sup, Std, Low-WL, Mod

- WL and Hi-WL mean supine rest, standing rest, low work load, moderate work load and high work load respectively. 136 Y, OHIRA et al .

consistently less than 7.4. The pH tended to decrease following exercise , but the only significant change was observed in the placebo group at day 16. There was also a transient decrease in pre-exercise blood pH at 7 days post-iron treatment .

DISCUSSION

It is clear that iron-deficient anemic humans and rats have lower maximal work

capacities than normal (1-8). Although it may appear that a reduction of O2 uptake capacity can explain the reduction in work capacity (7), this may not always be the

case. For example, the severely anemic subjects in this study tended to become exhausted prior to the point at which one normally reaches a maximal O2

consumption approximated by maximal heart rate (5,15). Also, when the subjects were asked why they stopped the treadmill exercise, a frequent cause was discomfort

in the leg muscles. Other physiological data lend some support to the idea that O2 consumption may not be the only or perhaps even the most critical limitation of anemic people to

perform work. Although the calculated O2 content was increased at 16th day, brachial venous blood PO2 and Hb-O2% saturation before and after exercise were

not significantly altered by the iron treatment. Initially, the subjects in our study had higher P50 values than normal suggesting a right-shifted O2-dissociation curve

due, in part, to elevated 2,3-DPG (Table 3) but not to PCO2 (Table 2) or pH (Table 5), which were within normal ranges. The 2,3-DPG value (ƒÊmol/g Hb) at 16 days

after iron treatment was significantly decreased from initial level and non-significant decrease was seen in P50.

Venous blood PCO2 was elevated in proportion to increases in Hb following iron treatment suggesting that the CO2 carrying capacity of blood is saturated. This

result is in agreement with the data reported by King and Mazal (14). They showed

a lower PCO2 in blood with lower hematocrit when the blood was exposed to

various levels of CO2 gas. In the current study, PCO2 levels had a significant correlation with Hb levels (r=0.44, p<0.01). Although CO2 is 20 times more soluble than O2, the red blood cell is essential for carrying, or converting to

bicarbonate, approximately 90% of the CO2 (20). Therefore, the ability to ade

quately clear CO2 from the working muscles because of reduced Hb level or red blood cell counts could be impaired in anemic individuals. This may lead to a reduced muscle pH which could limit the metabolic processes of skeletal mus cle (21), or may itself be responsible for "leg pain." Weinberg et al. (22,23) reported that iron-deficient anemic rats had less freezing (standing immobile for more than 10sec) and more rearing (front paws lifted off the floor) than controls. Cantwell (24) found that anemic children are less attentive and may be more hyperactive than normals. Although the basal metabolic rate measured in Sri Lankan individuals was not significantly correlated with Hb

(r=-0.25, p>0.05, unpublished data), the apparent increase in physical activity with anemia is supported by lactate changes. Severe iron deficiency anemia or iron

J. Nutr. Sci. Vitaminol. BLOOD GAS IN IRON DEFICIENCY ANEMIA 137

deficiency alone causes an elevation of resting blood lactate levels both in humans

(2) and rats (12). In fact, resting lactate levels in the current study were also higher than normal Sri Lankan subjects (approximately 0.6ƒÊmol/ml) with Hb levels greater than 13g/100ml (2). These findings suggest that iron deficiency with or without anemia causes an increase in tissue anaerobic glycolysis in some way. There was a significant positive correlation of r=0.46 (p<0.05, n=23) between Hb and myoglobin levels of vastus lateralis muscle samples obtained by needle biopsy from

Sri Lankan subjects (25). Decreased concentration and/or activities of iron containing substances which cause reduction of mitochondrial function were also found in iron-deficient rats (1, 3, 9-11, 26, 27). Such effects of tissue iron deficiency on mitochondrial function as well as the effect of anemia may cause the elevation of lactate even at rest. However, the aerobic metabolic capacity can be restored by iron treatment (3, 11, 26). The lower post-exercise lactate and higher PCO2 found at the

7th and 16th days after iron treatment might be caused by such improvement of mitochondrial oxidative phosphorylation changing the relative metabolism from anaerobic to aerobic.

As is shown in Table 5, heart rates during exercise at a given work load decreased following iron treatment. This may be due to increased O2 transport capacity of blood and/or increased O2 utilization capacity of tissue which is not directly related to Hb (6, 15, 28). Because no improvement was observed in the placebo group, this bradycardial effect following iron treatment was not due to the learning effect of repeated tests on the treadmill. Resting pH was maintained within normal range, except in one case, even though resting lactate was higher than normal (2). And the base excess, buffer base, and actual bicarbonate in the current study were low but tended to increase 16 days after iron treatment when aerobic metabolism probably was improved. Finch et al. (12) also found a lower base excess due to an elevated lactate maintaining a normal pH in severely iron-deficient and anemic rats. It is unclear why resting pH decreased significantly 7 days after iron treatment. However, the fact that two subjects had extremely low pH (7.277 and 7.299) with relatively higher lactate levels and elevated PCO2 could be the reason for the statistical significance. The possible reason why pH was not significantly low at the 16th day when PCO2 was elevated may be that the lactate levels were relatively low and that elevated Hb helped to buffer excess H+. Elevated blood lactate levels indicate the increase in the metabolic stress. The reason why the resting lactate was elevated 2 and 7 days after iron treatment is still unclear, but it is consistent with the previous study (15). Within 7 days after iron repletion, lactate elevation in response to the standard exercise was not significant. Although Jobsis and Stainsby (29) have shown that reduced O2 uptake is not an explanation for elevated venous blood lactate, Honig (30) has reported a model in which the non-uniformities of the PO2 within a muscle fiber could explain how a metabolically critical PO2 could occur even though mixed venous blood PO2 seems to be above a critical tension level for oxidative processes to proceed maximally

Vol. 29, No. 2, 1983 138 Y. OHIRA et al. within the muscle. An anemia-related elevation in blood lactate is a result of a greater dependence on anaerobic glycolysis. This may occur by a greater de pendence on the low oxidative muscle fibers because of the general pH induced depression of the metabolism in the more oxidatively dependent fibers, presumably used most in treadmill walking and slow running (31,32), or by the lack of a metabolically critical PO2 within the oxidative muscle fibers themselves.

This study was supported, in part, by the Nufeld Foundation and the J. B. Williams Co., Inc., New York, U. S.A. Authors also wish to thank the staff at Kandy General Hospital for their cooperation.

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