INTRODUCTION

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PREGNANCY-INDUCED CHANGES in respiratory control and acid-base regulation have been studied extensively in the resting state. These include increases in minute ventilation (E), tidal volume, and alveolar ventilation and a reduction in arterial PCO₂ (Pa_CO2) (1, 17, 25, 30). In accordance with conventional acid-base theory, these changes are accompanied by renal excretion of bicarbonate ([HCO₃]), resulting in a state of partly compensated respiratory alkalosis (arterial pH  7.43-7.47) (13, 17). These effects appear in the first trimester and may promote placental gas exchange before development of an effective fetal circulatory system (13).

Existing studies of acid-base balance during and after maternal exercise have reported conflicting results. Lehmann and Regnat (12) studied healthy pregnant women at rest and during 6 min of stationary cycling at both 50 and 80 W. They reported greater decreases in blood pH during exercise at both 50 and 80 W in pregnancy compared with measurements at 12 wk postpartum. Significantly lower absolute pH values were also encountered during the exercise at 80 W in pregnancy compared with the nonpregnant state. Conversely, Pivarnik et al. (25) reported higher arterial pH values in pregnant women at 37-wk gestation compared with postpartum during exercise bouts lasting 6 min on a cycle ergometer (50 and 75 W) and on a treadmill (67 m/min, 2.5% grade, and 67 m/min, 12% grade). Similar changes in pH in the transition from rest to exercise were reported under all exercise conditions in both groups, but [HCO₃] values decreased to a lesser extent in the pregnant group.

Lehman and Regnat (12) and Pivarnik et al. (25) employed the conventional Henderson-Hasselbalch approach to the study of acid-base balance in pregnancy and exercise. Unfortunately, this provides limited insight into the mechanisms controlling acid-base balance (6). In contrast, Stewart's physicochemical approach to acid-base analysis (26, 27) allows examination of the specific physicochemical determinants of changes in hydrogen ion concentration ([H⁺]) in individual fluid compartments. All variables are defined as independent or dependent, and all systems are assumed to behave in accordance with the principles of electoneutrality, conservation of mass, and dissociation equilibria. If the values for the independent variables {i.e., PCO₂, the strong ion difference ([SID]), and total weak acid ([A]_tot)} and dissociation constants are known, values for dependent variables ([H⁺], [HCO₃], dissociated weak acid, weak acid, carbonate, and hydroxide) can be determined mathematically. This approach to acid-base analysis has been validated in an animal model (23) and at rest and during recovery from exercise in young men (11, 14).

Only one study has used Stewart's approach to study acid-base regulation in healthy women. Kemp et al. (9) compared the responses of physically active pregnant women (mean gestational age, 33 ± 1 wk) to those of a nonpregnant control group in both the resting state and during recovery from a maximal cycle ergometer test. During pregnancy significantly lower values for [H⁺] in venous plasma in the resting state were the result of lower values for PCO₂ and [A]_tot (which would reduce [H⁺]). This effect was only partly offset by a lower [SID] (which would increase [H⁺]). Increases in [H⁺] in the transition from rest to maximal exercise were quantitatively similar in the two groups, and calculated contributions of PCO₂, [SID], and [A]_tot to changes in [H⁺] in the transition from rest to peak exercise, during early postexercise recovery (1-7 min postexercise), and during late postexercise (7-15 min) did not differ significantly in the pregnant vs. nonpregnant state. These findings suggested that pregnancy-induced changes in [H⁺] are established in the resting state and that exercise-induced changes in [H⁺] and its determinants are not affected by human pregnancy.

Unfortunately, the value of the study by Kemp et al. (9) to describe the effects of human pregnancy on [H⁺] and its independent determinants was limited by the use of venous plasma in the analyses. The composition of venous blood reflects the metabolic activity of the tissues that it drains, whereas that of arterial blood is consistent throughout the body. In this regard, Kemp et al. (9) studied venous blood drawn from the antecubital vein in the arm, whereas exercise was performed on a leg cycle ergometer. Clearly, the use of arterial (or arterialized) blood would have been more accurate and sensitive for analyzing changes in acid-base regulation induced by pregnancy and exercise.

The purpose to this study was to examine, by using Stewart's physicochemical approach, the effects of human pregnancy on arterialized plasma [H⁺] and its determinants in the resting state, as well as changes in [H⁺] in the transition from rest to both steady-state and non-steady-state exercise. It was hypothesized that the results would confirm the earlier findings of Kemp et al. (9) from venous blood that lower plasma [H⁺] in the resting state is the combined result of lower values for PCO₂ and [A]_tot and that this effect is partly offset by a lower [SID]. It was further hypothesized that changes in [H⁺] in the transition from rest to both steady-state and non-steady-state exercise would be similar in the pregnant vs. nonpregnant state and that the contributions of arterialized PCO₂ (Pa_CO2), [A]_tot, and [SID] to these changes would not differ significantly among groups.

	METHODS

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Subjects. Subjects were 15 healthy, nonsmoking, physically active pregnant women (pregnant group; PG). Results from the PG were compared with those of 15 healthy nonpregnant women with similar physical and demographic characteristics (control group; CG). Prospective subjects were recruited from local prenatal fitness classes and from the general population, via media advertisements, posters, flyers, and contact with local obstetricians. Medical clearance for pregnant subjects was obtained from the physician or midwife monitoring their pregnancy by using a standard form (Physical Activity Readiness Medical Examination for Pregnancy). Nonpregnant subjects completed the revised Physical Activity Readiness Questionnaire.

Exercise testing protocols. Subjects performed two exercise tests at least 1 day apart on a Sensor Medics (model 800S) constant-work-rate cycle ergometer. Subjects consumed a standard meal (350 kcal, 40% carbohydrate, 40% fat, 20% protein) 1-2 h before both tests and avoided strenuous physical activity and caffeine on the day of testing. The first test was used to determine the ventilatory threshold (T_vent) and to assess aerobic working capacity. The protocol involved 5 min of resting data collection and a 4-min warm-up at 20 W, followed by an increase in work rate of 20 W/min until a heart rate (HR) of 170 beats/min was reached (9, 16). Respiratory responses were measured on a breath-by-breath basis by using a computerized system (First Breath) that incorporates a respiratory mass spectrometer (Perkin-Elmer, MGA 1100) with a volume turbine (VMM-1100) as described by Hughson et al. (7). Breath-by-breath alveolar gas exchange was calculated by using the algorithm of Beaver et al. (3), and T_vent was identified by using the V-slope method (4). HR was monitored with both a Polar Vantage monitor and a Marquette Max-1 electrocardiograph. Oxygen pulse [O₂ uptake (O₂)/HR] at a heart rate of 170 beats/min was calculated as an index of aerobic working capacity (30).

Stewart's physicochemical analysis. Stewart's physicochemical equation (26, 27) was used to calculate [H⁺] by using values for the three independent variables (9, 11) and to calculate the contributions of the three independent variables to changes in [H⁺] in each group in response to exercise (9, 14)

14

11

11

7

Statistical analyses. Physical characteristics, T_vent, oxygen pulse at 170 beats/min, and measured changes in [H⁺] at the two exercise levels were compared between groups by using Student's t-statistics for independent samples. Data at rest and during exercise at 70 and 110% T_vent were compared within and between subjects by using a two-way ANOVA (groups vs. rest/exercise level) with repeated measures on the second factor. When a significant between-group main effect was observed, separate independent Student's t-statistics were used to identify significant differences among group means at rest, 70% Tvent, and 110% T_vent. When a significant within-subject main effect was observed, paired Student t-statistics were also used to detect significant differences among rest, 70% T_vent, and 110% T_vent within each group.

	RESULTS

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Subjects. Subjects in both groups were between 25 and 40 yr of age, and the mean values were 29.5 ± 0.9 and 26.9 ± 1.6 yr for the PG and CG, respectively (Table 1). Mean gestational age of the PG was 37.1 ± 0.2 wk. Within the CG, nine women were in the follicular phase of their menstrual cycle and six women were in the luteal phase. As expected, body mass and BMI were significantly higher in the PG compared with the CG at the time of testing. However, the PG's prepregnancy body mass and BMI were not different from those of the CG. There were no significant differences in mean age, body height, parity, O₂ at T_vent, and oxygen pulse (ml/beat) at 170 beats/min.

Ventilatory variables and HR. HR was significantly higher in the PG compared with the CG at rest and 70% T_vent, but the difference narrowed with increasing exercise intensity (Table 2). O₂ and the respiratory exchange ratio (RER) did not differ significantly between groups at rest or at either exercise level. E and the end-tidal PO₂ (PET_O2) were significantly higher in the PG at all measurement times. Ventilatory equivalents for oxygen (E/O₂) and carbon dioxide [E/CO₂ production (CO₂)] were significantly higher in the PG at all measurement times except for E/CO₂ at rest. The end-tidal PCO₂ (PET_CO2) was significantly lower in the PG at all measurement times and increased from rest to both work rates.

Dependent acid-base variables. During all three experimental conditions [H⁺] and [HCO₃] were lower in the PG vs. CG (Fig. 1, A and B). Results were statistically significant except for [H⁺] at 110% T_vent. [H⁺] increased significantly in the transition from rest to 70% T_vent and from 70 to 110% T_vent in both groups. [HCO₃] was significantly lower at 110% T_vent compared with at rest and 70% T_vent in both groups. Calculated mean changes in measured [H⁺] from rest to 70 and 110% T_vent were similar between groups (Fig. 2).

View larger version (2824K): [in this window] [in a new window]   Fig. 1.   A: measured hydrogen ion concentration ([H+]) at rest and at 2 work rates. B: calculated bicarbonate ion concentration ([HCO3]) at rest and at 2 work rates. * Significant difference between groups.  Significant change within group from rest. § Significant change within group from cycling at 70% ventilatory threshold (Tvent) to cycling at 110% Tvent, P < 0.05.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Changes in measured [H+]. Changes in measured [H+] from rest to both 70 and 110% Tvent were not significantly different between groups, P < 0.05.

Independent acid-base variables. Pa_CO2 was significantly lower in the PG vs. CG at rest and both exercise levels (Fig. 3). Pa_CO2 did not change significantly with exercise, except for an increase from rest to 70% T_vent in the PG. However, a trend for Pa_CO2 values to decrease from 70 to 110% T_vent was present in the CG. [SID] was significantly lower in the PG vs. CG at rest and 70% T_vent, but not at 110% T_vent (Fig. 4). There was a significant group × time interaction for [SID]. [SID] decreased significantly in the transition from rest to 110% T_vent within both groups and was significantly lower at 110 compared with 70% T_vent. [SID] increased significantly in the PG from rest to 70% T_vent.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Arterialized plasma PCO2 (PaCO2) at rest and at 2 work rates. * Significant difference between groups, P < 0.05.  Significant change within group from rest, P < 0.05.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Calculated plasma strong-ion difference ([SID]) at rest and at 2 work rates. * Significant difference between groups, P < 0.05.  Significant change within group from rest, P < 0.05. § Significant change within group from cycling at 70% Tvent to cycling at 110% Tvent, P < 0.05.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Total weak acid concentration ([A]tot) as reflected by total protein concentration ([TP]) at rest and at 2 work rates. * Significant difference between groups, P < 0.05.  Significant change within group from rest, P < 0.05. § Significant change within group from cycling at 70% Tvent to cycling at 110% Tvent, P < 0.05.

Relationship between measured and calculated [H⁺] values. Calculated [H⁺] values from Stewart's equation are compared with measured in Table 5. Calculated [H⁺] was not significantly different from measured [H⁺] at rest or during exercise in the CG. However, a small but statistically significant underestimation was present in the PG. Strong statistically significant correlations were also observed between measured and calculated [H⁺] in both groups. The only exception to this was the relationship at rest in the CG. This effect was attributable to the low variability of data in the resting state.

Contributions of independent variables to changes in [H⁺]. [SID] was the only variable to make a significantly different contribution to a change in [H⁺] between the groups (Fig. 6). At 70% T_vent an increase in [SID] in the PG attenuated the rise in [H⁺], whereas a decrease in [SID] in the CG contributed to the rise. At 110% T_vent, the contribution of [SID] to the rise in [H⁺] was significantly greater in the CG vs. PG. This appeared to be offset by a modest reduction in Pa_CO2 (P > 0.05) in the CG. This was reflected in a trend for a different contribution of Pa_CO2 between groups from rest to 110% T_vent, in which Pa_CO2 contributed to the rise in [H⁺] in the PG but attenuated the rise in [H⁺] in the CG.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Calculated contributions of independent variables to changes in [H+]. See text for explanation of significant differences between groups.

	DISCUSSION

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The central objective of this study was to examine, using the physicochemical approach of Stewart (26, 27), the effects of human pregnancy on mechanisms of acid-base regulation at rest and during exercise above and below T_vent. The two exercise intensities were chosen to examine the effects of pregnancy on acid-base regulation during moderate steady-state exercise and during strenuous non-steady-state exertion that represented a more severe challenge to maternal acid-base homeostasis. This study adds significantly to information available from the earlier study of Kemp et al. (9) because arterialized rather than venous plasma was employed for the analysis and the effects of exercise both below and above T_vent (i.e., steady-state and non-steady-state exercise conditions) were examined. It was hypothesized that acid-base balance would be significantly altered at rest in pregnancy and that absolute changes in [H⁺] in the transition from rest to exercise and the mechanisms responsible would be similar to the nonpregnant state.

Generalized metabolic and cardiorespiratory responses at rest and during both exercise tests and differences between groups were consistent with previous studies of exercising pregnant women. It is well documented that respiratory sensitivity to CO₂ is increased, resulting in higher values for E, E/O₂, and E/CO₂, as well as increased PET_O2 and reduced PET_CO2 both at rest and during submaximal exercise relative to the nonpregnant state (21, 22). RER does not appear to be altered at work rates below the onset of blood lactic acid accumulation (16, 29). It is well documented that HR is augmented both at rest and during submaximal exercise and that these differences in HR compared with the nonpregnant state decrease with increasing exercise intensity (21). Finally, although the effects of pregnancy on maximal aerobic power are controversial (29), existing studies indicate that T_vent is not changed (16, 30). Thus our results are consistent with and confirm the results of earlier studies of the effects of human pregnancy on generalized metabolic and cardiorespiratory responses to exercise.

Measured [H⁺] values in the PG were lower than in the CG at rest and at both exercise levels. Differences were statistically significant at rest and 70% T_vent, but significance was lost at 110% T_vent where the variability of measurements increased. Despite the difference in acid-base status at rest, both groups exhibited similar increases in [H⁺] in response to the acid-base balance challenge provided by exercise, as demonstrated by the lack of difference between groups in the measured changes in [H⁺] from rest to both work rates. These findings agree with those of Kemp et al. (9) and Pivarnik et al. (25) but differ from those of Lehmann and Regnat (12), who observed larger reductions in pH in pregnant subjects compared with nonpregnant controls in the transition from rest to submaximal exercise.

The present data also show that strenuous exercise in pregnancy does not produce high levels of [H⁺] in the maternal circulation that could have potentially detrimental effects on the fetus. The pH of the maternal blood at 110% T_vent (pH = 7.36) did not decrease to a greater extent than what would be expected in fetal venous blood at 37-wk gestation [pH = 7.36; (20)]. This suggests that the increases in the maternal [H⁺] with the exercise intensity above T_vent should not affect the fetal [H⁺] because changes in maternal [H⁺] are transient (9) and the maternal-fetal [H⁺] gradient was not reversed.

Values for [HCO₃] were lower in the PG vs. CG at rest and both exercise intensities. The decrease in [HCO₃] from rest to either exercise intensity was no greater in the PG than the CG. These results are in agreement with the earlier findings of Kemp et al. (9) from this laboratory but differ from those of Lehmann and Regnat (12), who found that exercise-induced decreases in base-excess were greater in pregnancy and those of Pivarnik et al. (25), who reported smaller exercise-induced decreases in [HCO₃].

In accordance with either Stewart's physicochemical approach or traditional acid-base analysis, significantly lower Pa_CO2 values in the PG vs. the CG would contribute to the maintenance of a lower [H⁺] in the PG at all measurement times. This confirms the importance of pregnancy-induced increases in respiratory sensitivity (2) to reduce Pa_CO2 and to maintain a lower [H⁺] than in the nonpregnant state. Augmented respiratory sensitivity in pregnancy has been attributed to increased circulating levels of progesterone, a known respiratory stimulant, and estrogen (5, 18). The increased estrogen level elevates the number of progesterone receptors in the hypothalamus (2, 5). In accordance with recent findings of Jennings and associates (8), the increase in E in pregnancy may also be due to changes in osmolality, [SID], and angiotensin II levels, which have been implicated in the control of ventilation. During pregnancy, osmolality decreases, [SID] decreases, and angiotensin II levels increase. In theory, all of these effects could contribute to the increase in E in pregnancy in concert with the effects of progesterone (28).

The lower [SID] in the PG compared with the CG at rest and both work rates resulted primarily from lower values for [Na⁺] and, to a lesser extent, [K⁺], in the PG, because there were no significant differences in [Cl] or [La] between groups. The reductions in [Na⁺] and [K⁺] may have been the result of pregnancy-induced hemodilution (or perhaps other mechanisms), because blood volume in pregnancy increases by 40-50% over nonpregnant levels (15, 24). Expansion of blood volume during pregnancy is attributable to an estrogen-mediated stimulation of the renin-angiotensin system, which, in turn, augments aldosterone secretion, as well as Na⁺ and water retention (15). Reduced [Na⁺] and [K⁺] values at rest in pregnancy have been observed previously (9, 17). As reported previously, pregnancy did not cause reductions in [Cl] (9, 18). This could be due to changes in renal absorption of Cl, ionic shifts between fluid compartments, or altered binding of Cl by plasma proteins.

The decrease in [SID] observed in both groups during the transition from rest to exercise at 110% T_vent was primarily the result of an increase in [La]. [La] did not differ significantly between groups at either exercise intensity, and increases were not significantly different between groups, although there have been reports of lower [La] for exercising pregnant women at work rates above T_vent (19, 30). Blunted [La] responses in pregnancy would, in theory, contribute to the maintenance of a lower [H⁺].

[A]_tot was lower, as a result of reductions in [Alb], in the PG vs. the CG at rest and both work rates, thus contributing to the lower [H⁺] in the PG. The lower [A]_tot in the PG was attributable to pregnancy-induced blood volume expansion and altered synthesis of proteins by the liver, as suggested by the decreased A/G ratio. The present results for [TP], [Alb], [Glob], and A/G ratio also agree with previous findings. [TP] and [Alb] decrease in pregnancy whereas [Glob] increases moderately (9). The overall result is a decrease in the A/G ratio (9, 24). [TP], and thus [A]_tot, increase during exercise, likely the result of exercise-induced hemoconcentration.

Similar absolute changes in [H⁺] in the transition from rest to 70 and 110% T_vent in the PG and CG suggested that the contributions of independent variables to the increase in [H⁺] in response to strenuous exercise were similar in the pregnant vs. nonpregnant state, as reported previously by Kemp et al. (9) in venous plasma. To examine this hypothesis further, Stewart's equation was utilized to calculate the contributions of the independent variables to changes in [H⁺] from rest to each work rate. The contribution of [A]_tot was not significantly different between groups at either exercise intensity. As discussed below, a trend (P = 0.07) was present for a different contribution of Pa_CO2 between groups at 110% T_vent. The contributions of [SID] to changes in [H⁺] were significantly different between groups at both exercise intensities, and a significant group × time interaction was observed for the absolute [SID] values. The altered behavior of [SID] in the pregnant state resulted in a negative contribution to the rise in [H⁺] at the 70% T_vent work rate and a smaller positive contribution to the [H⁺] rise at the 110% T_vent work rate. These findings are different from those of Kemp et al. (9), who found no pregnancy-induced effect on the contributions of any of the independent variables to changes in [H⁺] in recovery from a maximal exercise test. This discrepancy was attributable to the use of venous as opposed to arterial or arterialized plasma in this earlier study.

A trend for Pa_CO2 values to decrease from 70 to 110% T_vent was present in the CG (P = 0.06), but not in the PG (P = 0.32), suggesting that respiratory compensation for the rising [H⁺] was present in the CG at this work rate but not in the PG. The reduction in Pa_CO2 in the CG but not the PG could be easily explained if the rise in [H⁺] from 70 to 110% T_vent was greater in the CG vs. PG, but this was not observed. However, if [SID] rather than [H⁺] is the stimulus to chemoreceptors and ventilation as hypothesized by Jennings (8), the decrease in [SID] in the CG in the transition from rest to 110% T_vent (approximately twice that of the PG) would account for the differences in the behavior of Pa_CO2 at 110% T_vent.

Excellent agreement was observed between measured and calculated values for [H⁺] both at rest and during exercise in the CG and, except for values in the CG at rest (owing to low variability of measurements), strong statistically significant correlations were observed between measured and calculated [H⁺] in both groups under all experimental conditions. In accordance with earlier studies of exercising men (11), these results provide strong support for the validity and utility of Stewart's approach.

Within the PG, there was a small (2-3 meq/l) but statistically significant underestimation of [H⁺] using Stewart's equation and values for the independent variables. In accordance with modern acid-base theory (8), this could be an effect of changes in temperature, [SID],and/or osmolality on the dissociation constants used in Stewart's analysis. Indeed, our data showed significant differences in plasma osmolality and [SID] (but not temperature) in the pregnant vs. nonpregnant state. Given the small differences involved, it is unlikely that this introduced important mathematical errors in our calculation of contributions of the independent variables to changes in [H⁺]. It is also important to keep in mind that the purpose of using Stewart's approach is not to predict [H⁺] but to allow a more mechanistic approach to acid-base analysis than is possible by using the conventional Henderson-Hasselbalch approach by itself. The prediction of [H⁺] is usually done to show the validity of Stewart's approach and to provide assurance that no serious errors are involved in the measurement or calculation of the three independent variables. Taken by themselves, the results from the PG provide strong support for Stewart's approach on the basis of significant correlations between measured and calculated [H⁺]. Indeed, although the difference between the measured and calculated [H⁺] in the PG was significantly greater than in the CG, it was still quite small and in the same range observed in earlier validation studies (9, 11). Nevertheless, this effect should be considered in future studies of acid-base regulation in human pregnancy.

The present study confirmed earlier findings (1, 13, 17, 25, 30) that human pregnancy is accompanied by a partly compensated respiratory alkalosis as reflected by significantly reduced values for [H⁺], Pa_CO2, and [HCO₃] in arterialized plasma in the resting state. Application of Stewart's physicochemical approach was helpful in clarifying the involvement of metabolic, hepatic, and renal contributions to a reduction in [H⁺] in the resting state. The reduction of [A]_tot can be attributable, in part, to dilution of plasma proteins (and other weak acids) in an expanded maternal blood volume. However, a significant decrease in the A/G ratio also suggests altered synthesis of plasma proteins by the liver. Pregnancy-induced reduction in [SID] was the result of lower values for [Na⁺] and [K⁺], again presumably as a result of hemodilution. The reasons why [Cl] and [La] were not significantly reduced should be examined in future investigations of metabolic, renal, and ionic factors. The present study results also confirmed earlier findings that exercise-induced reductions in [H⁺] are quantitatively similar in the pregnant vs. nonpregnant state (9, 24). We also observed no significant differences between groups in the change in [HCO₃] in the transition from rest to either moderate (70% T_vent) or strenuous (110% T_vent) exercise. Application of Stewart's physicochemical approach demonstrated that there is a blunted [SID] response to exercise above and below T_vent in late pregnancy.

In conclusion, our results support our original hypothesis that lower [H⁺] of arterialized plasma in the resting state is the combined result of lower values for Pa_CO2 and [A]_tot and that this effect is partly offset by a lower [SID]. Whereas exercise-induced increases in [H⁺] are quantitatively similar in the pregnant vs. nonpregnant state, the mechanisms of adaptation from rest to exercise are different. The altered behavior of [SID] above and below T_vent in pregnancy is beneficial to maintain a lower [H⁺]. In addition, because there is a lesser contribution of [SID] to changes in [H⁺] induced by strenuous exercise in pregnancy, acid-base regulation may require less respiratory compensation at work rates above T_vent.