Dietary salt alters pulmonary function during exercise in exercise-induced asthmatics.

Epidemiological and experimental studies have suggested that dietary salt may play a role in airway responsiveness. We have previously shown that a low salt diet improves and a high salt diet exacerbates post-exercise pulmonary function in individuals with exercise-induced asthma. The aim of this study was to determine the influence of both elevated and restricted salt diets on pulmonary function during exercise in individuals with exercise-induced asthma. Nine men and six women participated in this double-blind, crossover study. The participants entered the study on their normal salt diet and were placed on either a low or high salt diet for 2 weeks. Each diet was randomized, with a 1 week washout period between diets before crossing over to the alternative diet for 2 weeks. The participants underwent treadmill testing at 85% of their age-predicted heart rate on the normal salt diet and at the end of each treatment period. Pulmonary function was assessed during exercise by arterial saturation (ear oximetry) and indirect calorimetry. Twenty-four hour urine collections confirmed compliance with the diets. Arterial saturation was reduced on the high and improved on the low salt diet at higher exercise intensities. Tidal volume and frequency selection during exercise varied with the diets, with a higher tidal volume and lower frequency on the high salt diet, but a lower tidal volume and higher frequency on the low salt diet. This suggested greater airway resistance during the high salt diet. In conclusion, the low salt diet improved and the high salt diet exacerbated pulmonary function during exercise in individuals with exercise-induced asthma. The mechanism of action remains unclear.

Dietary chloride as a possible determinant of the severity of exercise-induced asthma.

Dietary sodium chloride (NaCl) has been shown to alter the severity of exercise-induced asthma, but it is not known if the sodium and chloride ions have independent effects in this regard. The hypothesis tested in the present study was that both a low sodium, low chloride diet and a high sodium, low chloride diet would improve post-exercise pulmonary function in subjects with exercise-induced asthma (EIA) compared to a normal NaCl diet (NSD); but that neither of these diets would have an effect on post-exercise pulmonary function in control (non-EIA) subjects. Eight subjects who suffered from EIA and eight subjects who did not (control) took part in a double-blind crossover study. Pre- and post-exercise pulmonary function was assessed after 2 weeks on a NSD, a low NaCl diet (LSD, low sodium, low chloride) or a sodium bicarbonate diet (NaHCO3 diet, high sodium, low chloride). A 1 week washout period occurred between diets. Altering dietary sodium or chloride had no effect on pre-exercise (baseline) pulmonary function in either group or on post-exercise pulmonary function in control subjects. However, both the LSD and the NaHCO3 diet lessened the deterioration in post-exercise pulmonary function in EIA subjects. Comparing results from pre- to post-exercise, forced expiratory volume in 1 s (FEV1) at 15 min post-exercise differed significantly (P < 0.05) between diets [mean (SEM) 7 (4)% on the LSD, 14 (4)% on the NaHCO3 diet, and 19 (2)% on the NSD]. Similar patterns were observed for forced vital capacity (FVC), forced expiratory flow rate at 25%-75% FVC and peak expiratory flow rate. The NaHCO3 diet lessened the deterioration of post-exercise pulmonary function, but not to the extent of LSD. These data suggest that both sodium and chloride contribute to the worsening of EIA symptoms seen after consuming a normal or high NaCl diet.

Elevating dietary salt exacerbates hyperpnea-induced airway obstruction in guinea pigs.

Previous studies have indicated that increased dietary salt consumption worsens postexercise pulmonary function in humans with exercise-induced asthma (EIA). It has been suggested that EIA and hyperpnea-induced airway obstruction (HIAO) in guinea pigs (an animal model of EIA) are mediated by similar mechanisms. Therefore, the purpose of this study was to determine whether altering dietary salt consumption also exacerbated HIAO in guinea pigs. Furthermore, the potential pathway of action of dietary salt was investigated by blocking leukotriene (LT) production during HIAO in guinea pigs. Thirty-two male Hartley strain guinea pigs were split into two groups. One group (n = 16) of animals ingested a normal-salt diet (NSD) for 2 wk; the other group (n = 16) ingested a high-salt diet (HSD) for 2 wk. Thereafter, animals were anesthetized, cannulated, tracheotomized, and mechanically ventilated during a baseline period and during two dry gas hyperpnea challenges. After the first challenge, the animals were administered either saline or nordihydroguaiaretic acid, a LT inhibitor. Bladder urine was analyzed for electrolyte concentrations and urinary LTE(4). The HSD elicited higher airway inspiratory pressures (Ptr) than the NSD (P < 0.001) postchallenge. However, after infusion of the LT inhibitor and a second hyperpnea challenge, HIAO was blocked in both diet groups (P < 0.001). Nonetheless, the HSD group continued to demonstrate slightly higher Ptr than the NSD group (P < 0.05). Urinary LTE(4) excretion significantly increased in the HSD group compared with the NSD group within treatment groups. This study has demonstrated that dietary salt loading exacerbated the development of HIAO in guinea pigs and that LT release was involved in HIAO and may be moderated by changes in dietary salt loading.

Dietary salt restriction improves pulmonary function in exercise-induced asthma.

PURPOSE: Exercise-induced asthma (EIA) occurs in approximately 90% of persons with asthma. The mechanism has not been delineated. Epidemiological studies have suggested that dietary salt may play a role in airway responsiveness. Therefore, the purpose of this study was to determine the influence of both elevated and restricted salt diets on pulmonary function in subjects with EIA. METHODS: Eight subjects with EIA and eight subjects without EIA (control) participated in a double-blind crossover study. Pulmonary function was determined pre- and post-exercise challenge before and after 2 wk on a normal salt, sodium chloride, diet (NSD), a low salt diet (LSD), and a high salt diet (HSD). A 1-wk washout occurred between diets. RESULTS: Diet had no effect on preexercise pulmonary function values in either group and had no effect on postexercise pulmonary function values in control subjects. However, LSD improved and HSD worsened postexercise pulmonary function values in EIA subjects. Forced expiratory volume in 1 s (FEV1) decreased by at least 10% in EIA subjects with exercise. In EIA subjects, FEV1 decreased by 14+/-6% on LSD, 20+/-7% on NSD, and 24+/-6% on HSD at 15 min postexercise. Similar patterns were observed for forced vital capacity and peak expiratory flow rates. Although LSD did not normalize pulmonary function in EIA, it did improve it. CONCLUSIONS: These data suggest that individuals with EIA might benefit from lower salt diets.

Gender differences in tolerance to lower body negative pressure.

BACKGROUND: Previous studies suggest that women have lower tolerance than men do to lower body negative pressure (LBNP). The mechanism(s) responsible has not been determined. HYPOTHESES: Women would be less tolerant to presyncopal LBNP than men as determined by several indices of LBNP tolerance. Additionally, men and women, regardless of LBNP tolerance, would have similar cardiovascular responses to LBNP as presyncope was reached. METHODS: The subjects were 18 men and 18 women (average age 25) of similar fitness levels who volunteered for the study. A step-wise LBNP protocol to presyncope was employed. HR, stroke volume (SV), cardiac output (Q), BP, and systemic vascular resistance (SVR) were measured before and throughout the LBNP stress. Data from women were compared with those from all men, and to men with similar and higher LBNP tolerance. RESULTS: Women had significantly less LBNP tolerance than men regardless of index used: 30% less by duration of LBNP, 21% less by maximal LBNP tolerated, 44% less by a cumulative stress index, and 27% less by a linear tolerance index. Cardiovascular responses to LBNP were similar for women and men as presyncope was approached, whether the men were low-tolerant (LT) or high-tolerant (HT). In the 2 min pre-presyncope, HR increased by 80 +/- 6% in women, 72 +/- 7% in LT men and 96 +/- 14% in HT men; Q decreased by 47 +/- 3% in women, 52 +/- 6% in LT men and 55 +/- 2% in HT men. Similar comparisons occurred for the decline in BP and the rise in SVR. CONCLUSION: Women have lower LBNP tolerance than do men, although there is considerable gender overlap in tolerances. The cardiovascular response to LBNP is similar regardless of gender or tolerance level as presyncope is approached. Understanding the gender differences in LBNP tolerance may lie in determining how the LBNP stress is translated into a "trigger" for cardiovascular decompensation.

Hypertension in unilaterally nephrectomized rats induced by single-kidney transfection with angiotensinogen cDNA.

Plasmid expression vectors containing angiotensinogen (ATG) cDNA were complexed to cationic liposomes and injected into the renal artery of unilaterally nephrectomized rats to evaluate the effect of intrarenal ATG cDNA on arterial blood pressure and the renin-angiotensin system. Systolic blood pressures measured by tail cuff on days 12, 16, and 18 after transfection were significantly higher in rats that received ATG cDNA than in control rats that received the lac Z reporter gene. Plasma renin activity and plasma ATG concentration were unchanged. These results provide direct evidence that the availability of intrarenal ATG may be instrumental in the development of systemic hypertension.

Moderate sodium restriction does not alter lower body negative pressure tolerance.

HYPOTHESIS: Space travel with exposure to microgravity leads to a significant reduction in orthostatic tolerance on return to Earth, for which countermeasures are only partially successful. The purpose of this study was to examine the effect of moderate dietary sodium restriction on tolerance to LBNP. METHODS: Eight healthy men, age 25.1+/-1.3 yr, volunteered for the study. Subjects were exposed to presyncopal LBNP after consuming their "typical" diet (C) for 5 d and after consuming a sodium restricted (SR) diet for 5 d. Diet sequence was randomized and adherence was verified by 24-h urine collection on the 4th and 5th days of each diet. RESULTS: All subjects reached presyncope during the LBNP, regardless of diet. Urinary sodium excretion was 3390+/-950 mg on the C diet and 1174+/-560 mg on the SR diet. Urinary potassium was not different between the diets. Cumulative stress index scores were 655+/-460 (mm Hg x min) on the C diet and 639+/-388 (mm Hg x min) during SR. Cardiac volumes, BP and total peripheral resistance were not different at any stage of the LBNP between the diets, nor were catecholamines. Plasma renin activity, determined by radioimmunoassay, was significantly higher during SR at rest, and during all stages of LBNP in comparison with the control diet. CONCLUSION: Moderate dietary sodium restriction is not detrimental to orthostatic tolerance.

Cardiovascular effects of the breathhold used in determining pulmonary diffusing capacity.

BACKGROUND: The single-breath technique for determination of the diffusion capacity of the lung for CO (DlCO) requires a 10-s breathhold at total lung capacity. The assumption has been that this breathhold does not alter the components of DlCO, i.e., the diffusion capacity of the membrane (Dm) and the pulmonary capillary blood volume (Qc), and therefore measurement of these variables during breathhold represents these variables as they exist during normal breathing. The purpose of this study was to determine the effect of the 10-s breathhold on cardiac output (Q) and Qc while supine and standing. The hypothesis was that the standing posture would have a greater influence on Q and Qc during the breathhold than would the supine posture. METHODS: Twelve male subjects participated. Q, stroke volume (SV), heart rate (HR), BP (MAP), and total peripheral resistance (TPR) were determined before and during the 10-s breathhold determination of DlCO, Qc, and Dm. RESULTS: Results while supine were compared with those while standing. DlCO was reduced on standing, due mainly to a reduction in Qc. SV and Q decreased significantly during the 10-s breathhold in both postures. Both SV and Q decreased more when standing (-53% and -49.5%, respectively) than when supine (-40.5% and -36.5%, respectively). Thus, the 10-s breathhold caused significant reductions in Q and therefore may alter the measurement of DlCO and Qc. CONCLUSIONS: The greater decline in Q during the measurement of DlCO when standing would suggest that the DlCO and Qc values while breathing might be underestimated in the upright posture.

The heart is not necessarily empty at syncope.

BACKGROUND: Although extensively investigated, the mechanism(s) of post-spaceflight orthostatic intolerance has not been elucidated. Several researchers have proposed that the "trigger" for syncope is an empty ventricle, initiated when a hypercontractile state, possibly due to a sudden surge in epinephrine, causes the walls of the left ventricle to touch leading to a profound sympatho-inhibition and intense vagal stimulation. HYPOTHESIS: A markedly reduced left ventricular end systolic volume (LVESV) achieved during progressive, presyncopal-limited lower body negative pressure (LBNP) is the trigger for syncope. METHODS: Eight healthy men, age 25.1+/-1.3 yr, volunteered for the study. Changes in left ventricular end-diastolic volume and LVESV were measured, using two-dimensional echocardiography, at each stage of LBNP from rest up to presyncope (PS). Plasma venous catecholamine concentrations were measured at the end of each stage by high performance liquid chromatography (HPLC) with electrochemical detection. RESULTS: All subjects reached PS. Three men became bradycardic at presyncope while five remained tachycardic. LVESV decreased by 28% at PS with no evidence of ventricular cavity obliteration. Norepinephrine increased by 44% from rest to PS, but no epinephrine surge was detected (35% increase from rest to PS). CONCLUSION: These data indicate that it is possible to initiate syncope with only a 28% decrease in LVESV, and that sympatho-inhibition and bradycardia are not required elements for syncope to occur.

Validation of impedance cardiography during lower body negative pressure.

BACKGROUND: Both echocardiographic and impedance cardiographic techniques have been used individually for the determination of stroke volume (SV) during lower body negative pressure (LBNP). Impedance cardiography has not been validated during LBNP. HYPOTHESIS: The purpose of this study was to determine both the absolute values for SV and the change in SV for each stage of LBNP using both impedance and echocardiographic techniques during staged LBNP to presyncope. The hypothesis was that there would be no difference between the two techniques for either the absolute values of SV or for the change in SV with each stage of LBNP. METHODS: There were 16 men who volunteered to undergo LBNP. LBNP was lowered in 10 mmHg stages for 5 min per stage until presyncope was reached. Left ventricular SV was determined by two-dimensional echocardiography and impedance cardiography. Both the absolute values for SV and the change in SV from baseline at each stage of LBNP were compared for the two methods. RESULTS: There were no significant differences between the two techniques for the measurement of either the absolute SV or the change in SV with LBNP. The two methods correlated highly with r = 0.89 for the absolute SV values and r = 0.93 for the change in SV. Graphical analysis with the Bland-Altman analysis showed little bias in the impedance measurement for SV (-0.031 ml) and the change in SV (-2.7 ml). CONCLUSIONS: Impedance cardiography was a reliable measure of SV, as well as the change in SV, during LBNP stress to presyncope.

Bioelectric impedance as an index of thoracic fluid.

BACKGROUND: Thoracic electrical impedance (TEI) has been suggested as a means to monitor thoracic fluid changes. It was hypothesized that TEI would correlate significantly with an intrathoracic blood volume (pulmonary capillary blood volume) during postural shifts. METHODS: TEI was compared with pulmonary capillary blood volume (Vc) in six men and six women during postural stress. The diffusion capacity for carbon monoxide (DLCO) was used to determine Vc. Subjects were supine for 15 min and then stood quietly for 5 min. Measurements were made at the end of each period. Heart rate (HR), stroke volume (SV), and BP were determined for each posture. RESULTS: Women had higher TEI values at rest, confirming previous data. Cardiopulmonary variables change similarly for each gender on standing. TEI correlated significantly with Vc (p<0.01), and Vc was significantly correlated with SV (p<0.01). Total lung capacity and residual volumes were unchanged with posture. CONCLUSION: It is concluded that TEI can serve as a useful clinical and research monitor of thoracic fluid volume changes, even small volume changes, and correlates with a measured thoracic fluid compartment.

Physical activity, energy expenditure and fitness: an evolutionary perspective.

The model for human physical activity patterns was established not in gymnasia, athletic fields, or exercise physiology laboratories, but by natural selection acting over eons of evolutionary experience. This paper examines how evolution has determined the potential for contemporary human performance, and advances the experience of recently-studied hunter-gatherers as the best available (although admittedly imperfect) indicator of the physical activity patterns for which our genetically determined biology was originally selected. From the emergence of the genus Homo, over 2 million years ago (MYA), until the agricultural revolution of roughly 10000 years ago our ancestors were hunter-gatherers, so the adaptive pressures inherent in that environmental niche have exerted defining influence on human genetic makeup. The portion of our genome that determines basic anatomy and physiology has remained relatively unchanged over the past 40 000 years. Thus, the complex interrelationship between energy intake, energy expenditure and specific physical activity requirements for current humans remains very similar to that originally selected for Stone Age men and women who lived by gathering and hunting. Research investigating optimal physical activity for human health and performance can be guided by understanding the evolution of physical activity patterns in our species.

Pulmonary capillary blood volume during lower body negative pressure: effect of gender.

BACKGROUND: The purpose of this study was to examine the changes in pulmonary capillary blood volume (VC) in men and women during lower body negative pressure (LBNP). Additionally, the components of lung diffusion capacity were measured and evaluated for the effect of gender and LBNP. METHODS: There were 6 men and 6 women who underwent a staged LBNP protocol to -40 mm Hg. The diffusion of the lung for carbon monoxide (DLCO) was determined at two levels of inspired oxygen (21% and 60%). DLCO was separated into the diffusion capacity of the membrane (DLM) and the diffusion capacity of blood. RESULTS: During supine rest, DLCO values for men were larger than for women and were 39.8 +/- 3.8 and 32.2 +/- 2.2 ml.min-1.mm-1 Hg, respectfully. DLCO decreased equally with each stage of LBNP for men and women. VC during supine rest was greater for men (131 +/- 8 ml) than for women (92.7 +/- 7 ml). VC also declined with each stage of LBNP, and the decline was similar for men and women. DLM did not change with LBNP. CONCLUSION: The decrease in DLCO with LBNP is due to the reductions in thoracic blood volume in both men and women. These thoracic blood volume changes do not explain the previously reported reduced tolerances to LBNP for women because the reductions in thoracic blood volume were similar for men and women.

Women have lower tolerance to lower body negative pressure than men.

Studies of the cardiovascular response to lower body negative pressure (LBNP) in men and women have suggested that women may have less tolerance to LBNP than men, although tolerance per se was not determined. To investigate the effect of gender on tolerance to LBNP, 10 men 10 women were subjected to increasing levels of LBNP until presyncopal symptoms developed. The cumulative stress index (CSI) score was determined, as were cardiovascular variables. Women had 62% less tolerance to LBNP with a CSI of 412 +/- 43 mmHg/min compared with a CSI of 1,070 +/- 149 mmHg/min for men. Cardiovascular changes associated with LBNP were similar for men and women when expressed relative to the occurrence of presyncope, but women had a higher heart rate response when the data were expressed at absolute levels of LBNP (-30 and -50 mmHg LBNP). Thus men and women had similar cardiovascular adjustments to the LBNP, with the changes in women occurring lower levels of LBNP. These data are important in a consideration of the development of antigravitational countermeasures for women. These data raise questions as to the manner in which blood pools within the lower body in men and women under LBNP.

Cycling cadence alters exercise hemodynamics.

Previous studies on cycling cadence have focused on the economy of the cadence, in search of the optimal pedal cadence. The purpose of this study was to determine the hemodynamic changes associated with varying pedal cadence at a constant workload. It was hypothesized that increased pedal cadence would enhance the skeletal muscle pump, resulting in elevation of cardiac output. Seven cyclists were enlisted to cycle at 200 watts at pedal cadences of 70, 90 and 110 rpm (random order). Oxygen uptake, heart rate, stroke volume, cardiac output, blood pressure, and vascular resistance were determined. As has been previously shown, oxygen uptake increased with increased cadence (70, 90, 110 rpm) at this workload. Heart rate, stroke volume, cardiac output and blood pressure were increased, and vascular resistance decreased, with increased cadence. Cardiac output increased (34%) in excess of the increase in oxygen uptake (15%) as shown by the decrease (-14.5%) in the arterial-venous oxygen difference occurring with increasing cadence. Apparently, even though the workload was constant, the increase in pedal cadence resulted in a more effective skeletal-muscle pump which increased muscle blood flow and venous return. It is not known if this might contribute to the natural selection of higher cadences by cycling athletes, even though there is reduced economy.

Preconditioning with sodium deficits to improve orthostatic tolerance in rats.

We hypothesized that prior deficits in total body sodium would improve the subsequent ability of non-hypovolemic rats to maintain arterial pressure when subjected to an orthostatic challenge. This hypothesis was based on similarities in the response of neurohumoral cardiovascular control systems to lower-body negative pressure and negative sodium balance. Sodium deficits were induced in male Sprague-Dawley rats for 7-8 d by feeding sodium restricted diets, or by administering furosemide daily. After this, rats were allowed to regain a positive sodium balance for 1 d by increasing dietary intake or withholding furosemide, and receiving additional normal saline intraperitoneally. Rats subjected to these protocols had equal plasma volumes at the time they were anesthetized and evaluated for orthostatic tolerance. Furosemide-treated rats maintained a higher mean arterial pressure (MAP) than controls (70 +/- 10 vs 34 +/- 9 mm Hg) when rotated to a 90 degrees head-up position for 20 s. Rats receiving the lowest dietary sodium maintained the highest MAP (89 +/- 8 mm Hg) when placed at 60 degrees head-up for 5 min. Both before and during the orthostatic challenges, prior furosemide treatment was associated with a higher peripheral resistance, while prior dietary restriction of sodium was associated with a higher cardiac output. We conclude that preconditioning consisting of chronic sodium deficits can improve orthostatic tolerance in this animal model. The hemodynamic data indicate the different modes of preconditioning may have their primary effect on different determinants of orthostatic tolerance.

Difference in the cardiovascular response to prolonged sitting in men and women.

Orthostatic stress, sitting, results in adjustments of cardiovascular variables to maintain blood pressure and is prominent in a variety of occupations. Sitting serves as the control position for head-out water immersion studies. This study addressed gender differences in the cardiovascular response to prolonged sitting. Ten men and 10 women had cardiovascular measurements in the supine position compared with measurements during 2 hours in the seated position (Sit). Supine cardiovascular measurements were similar for both sexes. Heart rate changed similarly for both sexes with Sit. With Sit, men had elevated mean arterial pressure (9 +/- 3%), total peripheral resistance (54 +/- 9%), and decreased cardiac index (-27 +/- 5%), while women had no change in mean arterial pressure, lesser elevations in total peripheral resistance (17 +/- 7%), and lesser decreases in cardiac index (-12 +/- 5%) than men. Thus men, compared to women, had an elevated mean arterial pressure response to prolonged orthostatic stress.

Residual lung volume and ventilatory muscle strength changes following maximal and submaximal exercise.

In order to evaluate a mechanism which may be responsible for the often observed acute increase in residual lung volume (RV) following exercise, 12 non-smoking males (20-30 yrs) performed two bouts of exercise on separate days; one to maximal heart rate (HRmax) and one to 85% of HRmax for 20 min. Prior to exercise and at 5, 15, 30, 60 and 120 min post-exercise, the following parameters were measured: RV, forced vital capacity (FVC), forced expiratory volume (FEV1.0), forced expiratory flow (FEF75-85), maximal expiratory pressure (PEmax), and maximal inspiratory pressure (PImax). Significant (p < 0.05) increases occurred in RV at 5, 15 and 30 min following maximal exercise and at 5 and 30 min after submaximal exercise. Changes in RV between the two exercise bouts were generally greater (p < 0.05) for maximal exercise. Accompanying the increases in RV were significant (p < 0.05) decreases in PEmax and decreases in FVC, while FEV1.0 and FEF75-85 remained generally unchanged or were slightly elevated. The data suggest that decreases in expiratory muscle strength due to fatigue may in part be responsible for increases in RV.

Comparison of band and spot electrodes for the measurement of stroke volume by the bioelectric impedance technique.

OBJECTIVES: Conventional impedance cardiography uses a tetrapolar band electrode configuration to determine thoracic bioelectrical impedance changes and to quantify cardiovascular variables. The present study evaluated the effect of electrode type, band or electrocardiograph (spot), placed in the same anatomical location, on both the absolute value of cardiovascular variables and the percent change in the variable when the patient changed position from supine to standing. DESIGN: Prospective, randomized studies using repeated-measures design. SETTING: Research laboratory. PATIENTS: Twenty-one adult volunteer subjects. INTERVENTIONS: Subjects were studied while supine and again when standing, in random order as to first position. MEASUREMENTS AND MAIN RESULTS: Stroke volume and impedance-derived variables were measured. Values for stroke volume measured by the band electrode array were 85 +/- 7 and 49 +/- 3 mL for supine and standing, respectively. In contrast, stroke volume measured through the spot electrodes was 110 +/- 8 and 59 +/- 3 mL, respectively. These differences were due to a lower baseline thoracic bioelectrical impedance value measured through the spot electrodes. However, the percent changes for stroke volume with standing subjects were the same when measured through band (-41 +/- 3%) or spot (-44 +/- 3%) electrodes. CONCLUSIONS: The comparison of cardiovascular values obtained by impedance cardiography should be accomplished only when the values are obtained using similar electrode configuration and type. While there may be a variation in the absolute value of the variable, the direction and magnitude of the change in the variable is relatively unaffected by the electrode types as used in this study.