Gas Exchange during Exercise in Children with Thalassemia Major and Diamond-Blackfan Anemia

ABSTRACT: The two main goals of the study were: 1) to determine how O2 uptake, ventilation, and CO2 production during exercise were acutely affected by transfusion in children with congenital anemia (thalassemia major and Diamond-Blackfan syndrome) requiring hypertransfusion and chelation therapy and 2) to compare gas exchange responses to exercise of the anemic patients to normal values. Thirteen patients (age range 7-27) performed cycle ergometry with a progressively increasing work rate. Gas exchange was measured breath-by-breath. Tests were done before and after routine transfusion (mean increase in hematocrit 22%). The results were compared to 10 agematched normal children who performed the same protocol on two occasions separated by a 2-day interval, and to the reults of 109 normal children studied in this laboratory. Transfusion resulted in: 1) a small, but significant increase in the anaerobic threshold (9%) and 2) an increase in the slope of the relationship between O2-uptake and heart rates. Despite these improvements, the majority of the patients had abnormally low values of maximal O2 uptake, anaerobic threshold, and slope of the O2 uptake-heart rate relationship. The abnormalities were more marked in the older patients. Measurement of gas exchange during exercise may be helpful in determining an optimal hematocrit for patients on hypertransfusion regimens.

sufficient for normal cardiovascular and respiratory function at the same time that the consequent iron overload can be eliminated by chelation therapy. In addition, the presence of heart and lung impairment-known to occur in many of these patients (6-%)-may alter cardiorespiratory function to the extent that the "optimal" hematocrit may vary from patient to patient.
We reasoned that measurement of gas exchange during exercise could prove useful in these patients since it allows an assessment of the response of the cardiovascular and respiratory systems to increases in metabolic demand. The measurement of the dynamic responses of V02, VE, and VC02 (e.g. the anaerobic threshold) to exercise has not been frequently used to assess cardiac and respiratory function in children, but the ability of the organism to consume atmospheric O2 is the final and critical product of adjustments of cardiac output and ventilation. Thus, the analysis of gas exchange during exercise yields an insight into the adequacy of a patient's response that cannot be obtained from measurements of cardiac output or ventilation alone.
The present study was designed with two goals: I) to compare the gas exchange responses to exercise in children with congenital anemia to those in a large group of normal children previously studied in our laboratory (9,10) and 2) to examine how the gas exchange responses in the patients were acutely affected by transfusions. METHODS

Abbreviations
Pooulation. Thirteen ~atients. 1  Protocol. Exercise testing was done between 6 and 18 h before In patients with severe anemia such as thalassemia major and routine blood transfusion and between 18 and 36 h after transcongenital erythroid hypoplasia (Diamond-Blackfan syndrome), fusion (thereby ensuring a reequilibration of the intra and extrathe goal of therapy is to maintain an "optimal" hematocrit (1-vascular fluid volumes). The exercise protocol consisted of cycle 5). Ideally, this is a regimen in which frequency of transfusion is ergometry. Following a 3to Cmin period of unloaded ~edalling, a continually increasing (10 W/min) work rate ("rampx-pattern) 1216 COOPER ET AL. exercise in a single, brief test. Thus, long periods of exercise and the possibility of incurring a significant metabolic acidosis and prolonged fatigue were avoided. A mass spectrometer was used for rapid analysis of 02, CO2, and N2 concentration. Ventilation was measured either by a turbine volume transducer or by inspiratory and expiratory pneumotachographs and low resistance breathing valves. The electrical signals from these devices underwent analog-to-digitial computation for the on-line, breath-by-breath determination of V02 (STPD), VC02 (STPD), VE (BTPS); respiratory exchange ratio, ventilatory equivalent for 0 2 (VE/VO2) and C02 (VE/ VC02), and end-tidal partial pressures of O2 and C 0 2 as previously described (1 2). The data from each test were displayed online and stored on digital tape for subsequent analysis. The breath-by-breath data of each subject were interpolated at 1-s intervals, and moving averages could be obtained for smoothing of studies with a great deal of breath-to-breath variation.
Parameters ~f Gas Exchange Measured. V02max. This was the highest (or peak) V02 achieved by the subject. AT. The highest metabolic rate (work rate) for which the energy requirements may be obtained solely from O2 uptake, and, hence, without concomitant anaerobiosis. This was determined from gas exchange data by finding the V02 at which VE/VO2 increases without an increase in VE/VC02 (hyperventilation with respect to 02) (9, 1 1, 13).
Relationship between V02 and HR. This was an index of the cardiac response to the increasing metabolic requirement of exercise (10). HR was measured beat-by-beat using three anterior chest leads and the EKG was in continuous view via a high persistence EKG oscilloscope. Linear regression techniques were used to obtain the slope of the relationship between V 0 2 and HR (AV02/AHR) below the AT during the exercise protocol. Pre-and posttransfusion venous blood samples were obtained immediately prior to exercise for measurement of Hb concentration by standard techniques. Levels of 2,3-DPG were measured using an enzymatic method (Sigma Corp.). These measurements are included in Table 1. The mean increase in Hb concentration was 22%.
Control Group. To test for any possible systematic effects on gas exchange related to the study design (i.e. exercise on day 1 followed by exercise 2 days later), 10 healthy children were used as normal controls (mean age 1 1.8 yr, range 7-1 6 yr). They, like the anemic patients, had no previous experience in our exercise laboratory. Cycle ergometry testing was performed on day 1, then repeated on day 3. Similar to the anemic patients, no attempt was made to actively encourage the normal subjects to continue exercising at the higher work rates.

Normal Predicted Values of Gas Exchange Parameters. These
were obtained from a previous study in this laboratory in which cycle ergometry with ramp protocols was performed by 109 normal children ranging in age from 6 to 17 yr old (9,10). For the one subject aged 27 yr, predicted values were obtained from studies done with adults in this laboratory (14). Predicted values were based on each patient's body weight and gender. We used weight rather than age since many patients with thalassemia major have small body sizes for their age compared to normals, therefore, predicted values based on age would bias the interpretation of the results.
An activities questionnaire designed in this laboratory to subjectively assess levels of physical activity at home and at school was administered to the patients. The results were compared to those obtained in 109 normal children.
The independent t test was used to compare the results of the patients to those of the normal controls. To determine if the hypertransfusion had an effect on cardiorespiratory variables, an analysis of paired differences was made using the dependent t test. Statistical significance was taken at the p < 0.05 level.
Results are expressed as mean * 1 SD. For illustration of certain gas exchange variables, pretransfusion values in all 13 patients were superimposed and averaged (group-mean response) and compared to the posttransfusion values. This analysis is used as a noise-reducing technique for this type of data (16).

RESULTS
The results of exercise testing in the 10 control subjects were all within,the normal range of our laboratory. Prior to transfusion, the V02max values of the patients were significantly lower than normal (mean of 71 + 24% predicted) (Figs. 1 and 2).
Twelve of the 13 patients were below their predicted value and five were below 2 SD. (Consistent with this, the mean V02max of the patients was significantly lower than the mean V02max of the 10 control subjects.) There was no significant change in V02max in the patients following transfusion (mean pretransfusion V02max/kg was 28.6 + 9.8 ml Oz/min/kg; posttransfusion was 30.2 * 8.6 ml 02/min/kg) nor was there any change in the normal controls from day 1 to day 3. In addition, the patients achieved a mean maximal respiratory exchange ratio of 1.15 prior to transfusion and 1.18 following transfusion.
In contrast to the V02max, there was a small but significant increase in AT following transfusion. This occurred in 10 of 13 patients with a mean increase of 9 + 14% while no change was observed in the control group (Fig. 3). An example of this increase in AT is shown in one of the patients in Figure 4. Although the  values. There was no difference in \iozmax before and after transfusion the controls between days I and 3. In addition, AT in the patients wa:; in the patients or between the first and second visits in the normal below normal in the majority of patients both before and after transfu.. controls. sion. 12 18 COOPER increase in AT was greater in the patients aged 12 and younger (mean increase of 15%) compared to the seven older subjects (mean increase of 4%) those values did not differ significantly. The mean AT of the patients prior to transfusion was significantly lower than predicted, 75 f 16% (Fig. 2). All 13 patients were below their mean predicted and two were less than 2 SD of the normal. (Consistent with this, the mean AT of the patients was significantly lower than the mean AT of the 10 control subjects.) Despite the improvement after the transfusion, the mean AT of the anemic patients remained significantly lower than predicted (84 _+ 20%) with 1 1 of the 13 patients remaining below their predicted value.
Nine of the 13 patients had slopes of the V02-HR relationship which were below predicted and three were less than 2 SD (Fig.  2). The slope of the V02-HR relationship increased significantly in the patients following transfusion (nine of 13 increased; mean of 16%). This is consistent with the group mean response of HR and V 0 2 shown in Figure 5 demonstrating a relative tachycardia prior to transfusion. The change in the slope of the V02-HR relationship in response to the transfusion was significantly greater in the six children aged 12 and younger (mean increase of 36%) compared to the seven older children (mean increase of 13%).
There was no significant change in the levels of 2,3-DPG following transfusion. The mean pretransfusion level was 13.7 + 1.06 pmol/g Hb compared to the posttransfusion level of 13.6 k 2.3. These values were all within the normal range of nonanemic patients established in this laboratory (1 0-16 pmol/g Hb), but were considerably lower than expected for the degree of anemia. There was no correlation between the magnitude of the increase in AT and the change in levels of 2,3-DPG.
The activities questionnaire scores of the patients were indistinguishable from test values obtained in normal children. The patients' mean value was 105 k 32% predicted of the activity of normal children as assessed by the questionnaire. No predicted value was available for the 27-yr-old patient.

DISCUSSION
V02max and AT were low in the patients as a group both before and after transfusion, and severely so in a number of them (Figs. 1-3). This indicates that O2 availability is limited at lower than normal metabolic rates resulting in lactic acid production. Bufferring of the lactic acid by HC03-results in increased C 0 2 production relative to V02. The increase in VC02 causes a rise in V,, and forms the basis for the noninvasive determination of the AT (Fig. 4).
There are several possible explanations for the low AT and VO2max. First, a reduction in arterial O2 tension has been documented previously in patients with thalassemia major (7,8). Apparently, this is consequent to pulmonary vascular disease that develops in many of these patients (7,16,17). Second, the cardiomyopathy that often accompanies hemosiderosis may lead to a reduced cardiac output and insufficient O2 delivery to the working muscle cells during exercise. In fact, left ventricular dysfunction has recently been documented in asymptomatic patients with thalassemia with the technique of exercise radionuclide angiography (1 8). Third, anemia itself reduces arterial O2 content and this may affect 0 2 transport at the cellular level. Consistent with this are preliminary studies in this laboratory showing that normal subjects made anemic by phlebotomy have reductions in the AT (1 9).
Caution must be used in interpreting vo2max in patients because it is a highly effort-dependent parameter of aerobic exercise. A true V02max implies that a plateau of V 0 2 was achieved despite increasing work rate, but less than a third of normal children studied in this laboratory, who were actively encouraged to continue exercising at high work rates, actually achieved such a plateau (9). In the present study, neither the patients nor the control subjects were actively encouraged at higher work rates, and only one patient achieved a true plateau. Nonetheless, the maximal value for the respiratory exchange ratio in the patients was greater than 1.1 both pre-and posttransfusion suggesting that they closely approached, if not invariably achieved, a "true" V02max. In this context, one advantage of the AT is that its measurement does not require a maximal effort.
It is noteworthy that reduced AT and rapid heart rate response during exercise are also observed in adult patients with congestive heart disease (20). In these patients, the low AT reflects the insufficient availability of O2 in the periphery resulting from the reduced cardiac output. Our data provide evidence for a cardiac or pulmonary vascular impairment in some of the anemic patients.
Adaptation to reduced O2 carrying capacity of anemia can be mediated in three ways: by an increase in 2,3-DPG facilitating O2 unloading from hemoglobin (21), by increases in cardiac output, and by widening the arteriovenous O2 content difference. Levels of 2,3-DPG in the patients studied were low for the degree of anemia. This has been observed in patients with thalassemia major by other investigators (22). The etiology of this depressed 2,3-DPG response remains unclear. Correra el al. (23) have suggested that this lack of a red cell 2,3-DPG response may result in impaired O2 delivery and a "functional anemia." As a consequence, the patients were dependent solely on adjustments of Q and arteriovenous O2 content difference in order to maintain adequate O2 delivery to the tissues.
Our data demonstrate that the cardiac adaptation for the anemia was a more rapid HR response to exercise (Fig. 5), as has been observed in subjects with anemia of other etiologies (24)(25)(26). This ability to adapt to the anemic state may explain why the increase in AT was small (9%) compared to the increase in Hb (22%) following transfusion. Moreover, in the older patients, whose HR during exercise were generally higher than normal both pre-and posttransfusion, the effect of transfusion was smaller than in the younger patients. This suggests that adaptation to anemia is diminished in the presence of heart or pulmonary vascular disease.
Transfusion improved the AT (Fig. 3) and the V 0 2 -H~ relationship in the patients. The increase in AT was most likely due to the increase in arterial 0 2 content following transfusion. This would result in higher p 0 2 in the muscle tissue and facilitate O2 transport into the cells. These observations are consistent with several previous studies in the literature. Thomson et al. (24) found improved O2 transport attributed to increases in arterial O2 tension following reinfusion of blood in normal subjects.
Miller et al. (25) demonstrated increased anaerobic metabolism (excessive lactic acidemia) during exercise in patients with sicklecell anemia. Finally, Woodson et al. (26) showed that V,/VO2 was higher in healthy subjects made anemic by phlebotomy. The lack of increase in the VO2max in our patients may be explained by our not "pushing" the subjects at the higher work rates.
Despite the increase in AT following transfusion, both the V0,max and the AT were significantly less than predicted (Figs  1-3). Only two of the patients had values of the AT greater than predicted following transfusion. Thus, a mild abnormality in gas exchange during exercise seems to be present even in some of the younger patients. In contrast, the self-assessment of physical activity in the patients was comparable to normal children of the same age and gender. The discrepancy between this and the results of the exercise studies suggests several possible explanations. First, the physiologic abnormalities that we observed might not have been severe enough to inhibit the child's "natural" inclination for physical activity. If so, the cardiorespiratory and metabolic adaptations to normal levels of physical activity are impaired in the patients. Second, the self-assessment done by patients who undergo frequent hospitalizations and nightly chelation therapy might be inaccurate, and, in truth, their level of physical activity is below normal. Alternatively, the questionnaire may have accurately described the time spent by the patients in physical activity, but the actual work done by them during activity may have been less than in normal children.
The V02max, AT, and relationship between V02 and HR deteriorated with increasing age (Fig. 2). In addition, the effect of the transfusion on the V02-heart rate slope was significantly less in the older patients. These data suggest that either the disease process itself, or the effects of iron overload in the myocardium or lungs (especially in the older patients who had not benefited from chelation therapy as early in their lives as had the younger subjects) results in progressive impairment of cardiorespiratory function in response to exercise. The noninvasive measurements described herein can be useful in the continuing evaluation of hypertransfusion and chelation therapy.