Respiratory Abstracts 2

© 2012

Central neural mechanisms of progesterone action: application to the respiratory system

            (Bayliss and Millhorn 1992) Download

Around the turn of the century, it was recognized that women hyperventilate during the luteal phase of the menstrual cycle and during pregnancy. Although a causative role for the steroid hormone progesterone in this hyperventilation was suggested as early as the 1940s, there has been no clear indication as to the mechanism by which it produces its respiratory effects. In contrast, much mechanistic information has been obtained over the same period about a different effect of progesterone, i.e., the facilitation of reproductive behaviors. In this case, the bulk of the evidence supports the hypothesis that progesterone acts via a genomic mechanism with characteristics not unlike those predicted by classic models for steroid hormone action. We recently, therefore, undertook a series of experiments to test predictions of those same models with reference to the respiratory effects of progesterone. Here we highlight the results of those studies; as background to and precedent for our experiments, we briefly review previous work in which effects of progesterone on respiration and reproductive behaviors have been studied. Our results indicate that the respiratory response to progesterone is mediated at hypothalamic sites through an estrogen- (E2) dependent progesterone receptor- (PR) mediated mechanism requiring RNA and protein synthesis, i.e., gene expression. The E2 dependence of the respiratory response to progesterone is likely a consequence of the demonstrated induction of PR mRNA and PR in hypothalamic neurons by E2. In short, we found that neural mechanisms underlying the stimulation of respiration by progesterone were similar to those mediating its reproductive effects.

Estrogen replacement therapy prevents airway dysfunction in a murine model of allergen-induced asthma

            (Dimitropoulou, Drakopanagiotakis et al. 2009) Download

We previously reported that 17beta-estradiol (E2) prevents hyperresponsiveness to carbachol of murine asthmatic tracheal rings in vitro. We now investigated whether E2 is similarly effective in reducing airway hyperreactivity in a murine model of allergic asthma in vivo. Female ovariectomized BALB/c mice were rendered asthmatic by a 25-day protocol of sensitization to ovalbumin. Under positive-pressure ventilation, anesthetized asthmatic mice exhibited a dramatic increase in airway responsiveness to increasing doses of inhaled methacholine compared to PBS-sensitized controls, as reflected in decreased dynamic compliance of the respiratory system and increased tissue damping, tissue elastance, and airway resistance. Furthermore, asthmatic mice exhibited hypercellularity and increased protein concentration in the bronchoalveolar lavage, strong signs of peribronchial cuffing with inflammatory cells and increased goblet cell activity. To test the effects of estrogen, three additional groups of mice were implanted subcutaneously with different amounts of slow-release E2 pellets at the time of ovariectomy and rendered asthmatic as before. E2 dose-dependently inhibited airway hyperresponsiveness to methacholine, reduced bronchoalveolar lavage hypercellularity, and virtually eliminated histologic signs of inflammation and goblet cell hyperactivity. The inflammation and airway hyperactivity in asthmatic mice was associated with an increase in bronchoalveolar lavage levels of TGFbeta1, which was completely abolished in E2-treated asthmatic mice. We conclude that estrogen replacement therapy effectively ameliorates the pathologic profile of murine allergic asthma.

17beta-estradiol protects the lung against acute injury: possible mediation by vasoactive intestinal polypeptide

            (Hamidi, Dickman et al. 2011) Download

Beyond their classical role as a class of female sex hormones, estrogens (e.g. 17beta-estradiol) exert important biological actions, both protective and undesirable. We have investigated the ability of estradiol to protect the lung in three models of acute injury induced by 1) oxidant stress due to the herbicide paraquat; 2) excitotoxicity, caused by glutamate agonist N-methyl-d-aspartate; and 3) acute alveolar anoxia. We also assessed the role of estrogen receptors (ER) ERalpha and ERbeta and the neuropeptide vasoactive intestinal peptide (VIP) in mediating this protection. Isolated guinea pig or rat lungs were perfused in situ at constant flow and mechanically ventilated. The onset and severity of lung injury were monitored by increases in pulmonary arterial and airway pressures, wet/dry lung weight ratio, and bronchoalveolar lavage fluid protein content. Estradiol was infused into the pulmonary circulation, beginning 10 min before induction of injury and continued for 60-90 min. Lung injury was marked by significant increases in the above measurements, with paraquat producing the most severe, and excitotoxicity the least severe, injury. Estradiol significantly attenuated the injury in each model. Both ER were constitutively expressed and immunohistochemically demonstrable in normal lung, and their selective agonists reduced anoxic injury, the only model in which they were tested. As it protected against injury, estradiol rapidly and significantly stimulated VIP mRNA expression in rat lung. Estradiol attenuated acute lung injury in three experimental models while stimulating VIP gene expression, a known mechanism of lung protection. The up-regulated VIP expression could have partially mediated the protection by estrogen.

Testosterone levels in men with chronic obstructive pulmonary disease with or without glucocorticoid therapy

            (Kamischke, Kemper et al. 1998) Download

Under the clinical impression that patients with chronic obstructive pulmonary disease (COPD) may demonstrate signs compatible with hypogonadism, we investigated whether oral glucocorticoid therapy is associated with testosterone deficiency. Thirty six men with COPD of whom 16 were receiving oral glucocorticoid medication (mean+/-SEM dose 9.4+/-1.1 mg prednisolone) were investigated in a cross-sectional cohort study. Patients with or without oral glucocorticoid therapy were not different in terms of age, smoking history and additional therapy. Vital capacity, forced expiratory volume in one second, airway resistance, intrathoracic gas volume and blood gases at rest were not different between the groups. However, patients receiving glucocorticoids had a shorter 6 min walking distance (mean+/-SEM 205+/-27 versus 288+/-26 m; p=0.02) compared to patients without oral steroid therapy. Serum levels of testosterone (mean+/-SEM 13.7+/-0.9) were below normal (<12 nM) in 15 of 36 patients. Serum testosterone did not correlate with any other evaluated parameter. Serum levels of free testosterone (free T) (mean+/-SEM 172.3+/-7.8 pM) were decreased in 25 of the 36 patients, including all patients receiving glucocorticoid treatment. In the 16 patients taking glucocorticoids free T was correlated (p=0.016) with the current glucocorticoid dosage (r=-0.504; p=0.007) and the body mass index (r=0.241; p=0.037). All other parameters examined revealed no significant correlations in multiple regression analysis. Glucocorticoid treatment appears to aggravate hypogonadism and a therapeutic study using testosterone in patients with chronic obstructive pulmonary disease receiving glucocorticoid medication appears warranted.

The effects of hormone replacement therapy on sleep-disordered breathing in postmenopausal women: a pilot study

            (Manber, Kuo et al. 2003) Download

STUDY OBJECTIVES: To evaluate the impact of estrogen and estrogen plus progesterone hormone-replacement therapy (HRT) on mild-to-moderate sleep-disordered breathing (SDB) in postmenopausal women. DESIGN AND SETTING: Within-subjects, progesterone placebo-controlled prospective HRT trial in a clinical laboratory. PARTICIPANTS: Six postmenopausal women, diagnosed with mild-moderate SDB. INTERVENTION: Transdermal estradiol and oral micronized progesterone. MEASUREMENTS AND RESULTS: Subjects underwent polysomnography (PSG) on four occasions: a screening/adaptation night; a baseline night on no HRT; and two nights on HRT: one night after 7 to 12 days on estrogen plus placebo followed by a second night after 7-13 days on estrogen plus progesterone. The PSG was performed with a Sandman computerized PSG system using a standard clinical montage. Modified sleep diaries were used in the baseline week and throughout the study period. Mood was measured with the 20-item version of the Positive and Negalive Affect Schedule (PANAS). Estrogen monotherapy was associated with a significant reduction in the overall apnea-hypopnea index (AHI) (from a mean of 22.7 events per hour at baseline to a mean of 12.2 events per hour), but the AHI reduction on estradiol plus progesterone relative to baseline was not statistically significant (AHI=16.2 events per hour). Similar results were found for the percentages of total sleep time and of total non-rapid eye movement sleep time with oxygen saturation less than 90%. Estrogen, neither alone nor in combination with progesterone, significantly altered PSG- or diary-based measures of total sleep time, time to sleep onset, or time awake after sleep onset. CONCLUSIONS: While the data are preliminary and based on a small number of subjects, estrogen appeared to have a substantial beneficial effect on measures of SDB in postmenopausal women. Overall, no additional benefit was seen with the addition of progesterone. In fact, progesterone attenuated the beneficial effects of estrogen in 4 out of the 6 participants.

Estrogen regulates pulmonary alveolar formation, loss, and regeneration in mice

            (Massaro and Massaro 2004) Download

Lung tissue elastic recoil and the dimension and number of pulmonary gas-exchange units (alveoli) are major determinants of gas-exchange function. Loss of gas-exchange function accelerates after menopause in the healthy aged and is progressively lost in individuals with chronic obstructive pulmonary disease (COPD). The latter, a disease of midlife and later, though more common in men than in women, is a disease to which women smokers and never smokers may be more susceptible than men; it is characterized by diminished lung tissue elastic recoil and presently irremediable alveolar loss. Ovariectomy in sexually immature rats diminishes the formation of alveoli, and estrogen prevents the diminution. In the present work, we found that estrogen receptor-alpha and estrogen receptor-beta, the only recognized mammalian estrogen receptors, are required for the formation of a full complement of alveoli in female mice. However, only the absence of estrogen receptor-beta diminishes lung elastic tissue recoil. Furthermore, ovariectomy in adult mice results, within 3 wk, in loss of alveoli and of alveolar surface area without a change of lung volume. Estrogen replacement, after alveolar loss, induces alveolar regeneration, reversing the architectural effects of ovariectomy. These studies 1) reveal estrogen receptors regulate alveolar size and number in a nonredundant manner, 2) show estrogen is required for maintenance of already formed alveoli and induces alveolar regeneration after their loss in adult ovariectomized mice, and 3) offer the possibility estrogen can slow alveolar loss and induce alveolar regeneration in women with COPD.

Menopausal estrogen therapy predicts better nocturnal oxyhemoglobin saturation

            (Saaresranta, Polo-Kantola et al. 2006) Download

OBJECTIVES: The respiratory responses in the few previous studies evaluating the effects of short-term unopposed estrogen therapy on breathing in postmenopausal women have been inconsistent. We performed a study to investigate whether long-term estrogen therapy would prevent age-related decline in nocturnal arterial oxyhemoglobin saturation and whether higher serum estradiol concentration is associated with better arterial oxyhemoglobin saturation. METHODS: Sixty-four healthy postmenopausal women were followed-up for 5 years in a 5-year prospective open follow-up study. The women were users or non-users of estrogen therapy according to their personal preference. RESULTS: Mean overnight arterial oxyhemoglobin saturation was similar at baseline (94.3 +/- 1.1%) and after follow-up (94.5 +/- 1.6%). Present estrogen users had higher mean arterial oxyhemoglobin saturation (95.2 +/- 1.4%) than present non-users (94.0 +/- 1.5%), when adjusted for age and body mass index (p = 0.042). The change in mean arterial oxyhemoglobin saturation during follow-up was not associated with serum estradiol concentration at baseline but associated with estradiol at follow-up (p = 0.042), when adjusted for age and body mass index. At follow-up, women with higher serum estradiol concentration had also higher mean nocturnal arterial oxyhemoglobin saturation (Pearson r = 0.29, p = 0.019) and lower apnea-hypopnea index (Spearman r = -0.28, p = 0.031). The pooled current estrogen users spent proportionally less time with SaO(2) below 90% than non-users (ANCOVA adjusted for age and BMI, p = 0.017). CONCLUSIONS: Estrogen use and especially high serum estradiol concentration predict higher mean overnight arterial oxyhemoglobin saturation. The present data suggest that estrogen therapy has favorable respiratory effects.

Anabolic steroids in COPD: a review and preliminary results of a randomized trial

            (Sharma, Arneja et al. 2008) Download

Patients with severe chronic obstructive pulmonary disease (COPD) commonly develop weight loss, muscle wasting, and consequently poor survival. Nutritional supplementation and anabolic steroids increase lean body mass, improve muscle strength, and survival in patients enrolled in comprehensive rehabilitation programs. Whether anabolic steroids are effective outside an intensive rehabilitation program is not known. We conducted a prospective, double-blind, placebo-controlled, 16-week trial to study the benefits of anabolic steroids in patients with severe COPD who did not participate in a structured rehabilitation program. Biweekly intramuscular injections of either the drug (nandrolone decanoate) or placebo were administered. Sixteen patients with severe COPD were randomized to either placebo or nandrolone decanoate. The placebo group weighed 55.32 +/- 11.33 kg at baseline and 54.15 +/- 10.80 kg at 16 weeks; the treatment group weighed 68.80 +/- 6.58 at baseline and 67.92 +/- 6.73 at 16 weeks. Lean body mass remained unchanged, 71 +/- 6 vs. 71 +/- 7 kg in placebo group and 67 +/- 7 vs. 67 +/- 7 in treatment group, at baseline and 16 weeks respectively. The distance walked on 6 min was unchanged at baseline, 8 weeks, and 16 weeks in placebo (291.17 +/- 134.83, 282.42 +/- 115.39, 286.00 +/- 82.63 m) and treatment groups (336.13 +/- 127.59, 364.83 +/- 146.99, 327.00 +/- 173.73 m). No improvement occurred in forced expiratory volume in one second, forced vital capacity, maximal inspiratory pressure, maximal expiratory pressure, VO(2) max or 6-min walk distance or health related quality of life. Administration of anabolic steroids (nandrolone decanoate) outside a dedicated rehabilitation program did not lead to either weight gain, improvement in physiological function, or better quality of life in patients with severe COPD.

Characterization of the vasodilatory action of testosterone in the human pulmonary circulation

         (Smith, Bennett et al. 2008) Download

AIM: To assess for the first time the vasodilatory effect of testosterone in the human pulmonary circulation utilizing both isolated human pulmonary arteries and isolated perfused human lungs. In addition, a secondary aim was to determine whether there was any difference in the response to testosterone dependent upon gender. METHODS: Isolated human pulmonary arteries were studied by wire myography. Vessels were preconstricted with U46619 (1 nM-1 microM) prior to exposing them to either testosterone (1 nM-100 microM) or ethanol vehicle (<0.1%). Isolated lungs were studied in a ventilated and perfused model. They were exposed to KCl (100 mM), prior to the addition of either testosterone (1 nM-100 microM) or ethanol vehicle (<0.1%). RESULTS: Testosterone caused significant vasodilatation in all preparations, but a greater response to testosterone was observed in the isolated perfused lungs, 24.9 +/- 2.2% at the 100 microM dose of testosterone in the isolated pulmonary arteries compared to 100 +/- 13.6% at the 100 microM dose in the isolated perfused lungs. No significant differences in the response to testosterone were observed between sexes. CONCLUSION: Testosterone is an efficacious vasodilator in the human pulmonary vasculature and this is not modulated by patient sex. This vasodilator action suggests that testosterone therapy may be beneficial to male patients with pulmonary arterial hypertension.

Reduced pulmonary function is associated with lower levels of endogenous total and free testosterone. The Tromso study

            (Svartberg, Schirmer et al. 2007) Download

Men with chronic obstructive pulmonary disease have reduced endogenous testosterone levels. Little is known, however, about the relationship between pulmonary function and endogenous testosterone levels in a general population. In the present study we have examined the cross-sectional associations between sex hormones measured by immunoassay and pulmonary function assessed with spirometry and oxygen saturation in 2,197 men participating in the fifth Tromso study. The data were analyzed by univariate correlations, multiple linear regression analyses and analyses of variance and covariance. Total and free testosterone were positively and independently associated with forced vitality capacity, FVC (% of predicted) (P = 0.001 and P = 0.006, respectively) and forced expiratory volume in 1 second, FEV(1 )(% predicted) (P = 0.033 and P = 0.002, respectively), and men with severe pulmonary obstruction (FEV(1) % of predicted < 50) had lower free testosterone levels (P = 0.005). In this cross-sectional data from Tromso, a reduction in pulmonary function was associated with lower levels of total and free testosterone. We suggest that the reduction of total and free testosterone could be due to an alteration of the hypothalamic-pituitary response.

Estrogens and development of pulmonary hypertension: interaction of estradiol metabolism and pulmonary vascular disease

            (Tofovic 2010) Download

Severe pulmonary arterial hypertension (PAH) is characterized by clustered proliferation of endothelial cells (ECs) in the lumina of small size pulmonary arteries resulting in concentric obliteration of the lumina and formation of complex vascular structures known as plexiform lesions. This debilitating disease occurs more frequently in women, yet both animal studies in classical models of PAH and limited clinical data suggest protective effects of estrogens: the estrogen paradox in pulmonary hypertension. Little is known about the role of estrogens in PAH, but one line of evidence strongly suggests that the vascular protective effects of 17beta-estradiol (estradiol; E2) are mediated largely by its downstream metabolites. Estradiol is metabolized to 2-hydroxyestradiol (2HE) by CYP1A1/CYP1B1, and 2HE is converted to 2-methoxyestradiol (2ME) by catechol-O-methyl transferase. 2ME is extensively metabolized to 2-methoxyestrone, a metabolite that lacks biologic activity, but which may be converted back to 2ME. 2ME has no estrogenic activity, and its effects are mediated by estrogen receptors-independent mechanism(s). Notably, in systemic and pulmonary vascular ECs, smooth muscle cells, and fibroblasts, 2ME exerts stronger antimitotic effects than E2 itself. E2 and 2ME, despite having similar effects on other cardiovascular cells, have opposing effects on ECs; that is, in ECs, E2 is promitogenic, proangiogenic, and antiapoptotic, whereas 2ME is antimitogenic, antiangiogenic, and proapoptotic. This may have significant ramifications in severe PAH that involves uncontrolled proliferation of monoclonal apoptosis-resistant ECs. Based on its cellular effects, 2ME should be expected to attenuate the progression of disease and provide protection in severe PAH. In contrast, E2, due to its mitogenic, angiogenic, and antiapoptotic effects (otherwise desirable in normal quiescent ECs), may even adversely affect endothelial remodeling in PAH, and this may be even more significant if the E2's effects on injured endothelium are not opposed by 2ME (eg, in the event of reduced E2 conversion to 2ME due to hypoxia, inflammation, drugs, environmental factors, or genetic polymorphism of metabolizing enzymes). This review focuses on the effects of estrogens and their metabolites on pulmonary vascular pathobiology and the development of experimental PAH and offers potential explanation for the estrogen paradox in PAH. Furthermore, we propose that unbalanced estradiol metabolism may lead to the development of PAH. Recent animal data and studies in patients with PAH support this concept.

Hypogonadism, quadriceps weakness, and exercise intolerance in chronic obstructive pulmonary disease

         (Van Vliet, Spruit et al. 2005) Download

RATIONALE: Circulating levels of testosterone and gonadotrophins of patients with chronic obstructive pulmonary disease (COPD) have never been compared with those of elderly men with normal pulmonary function. Moreover, the relationship of hypogonadism with quadriceps muscle weakness and exercise intolerance has been studied scarcely in men with COPD. OBJECTIVES: To compare circulating levels of hormones of the pituitary-gonadotrophic axis of men with COPD with those of age-matched control subjects. Moreover, to study the relationship of hypogonadism with quadriceps muscle force, 6-min walking distance, and systemic markers of inflammation in the patients. METHODS AND MEASUREMENTS: Circulating levels of follicle-stimulating hormone, luteinizing hormone, testosterone, and sex hormone-binding globulin were determined, and free testosterone was calculated in 78 patients (FEV1: 44 +/- 17% of the predicted values) and 21 control subjects. Moreover, quadriceps muscle force, 6-min walking distance, number of pack-yr, and systemic inflammation were determined. MAIN RESULTS: Follicle-stimulating hormone and luteinizing hormone were higher in the patients, whereas testosterone was lower (p < or = 0.05). The latter finding was also present in 48 non-steroid-using patients with normal blood gases. Low androgen status was significantly related to quadriceps muscle weakness (r = 0.48) and C-reactive protein (r = -0.39) in the patients, but not to exercise intolerance, the number of pack-yr, or increased circulating levels of interleukin 8 or soluble receptors of tumor necrosis factor alpha. CONCLUSIONS: In contrast to exercise intolerance, quadriceps muscle weakness is related to low circulating levels of testosterone in men with COPD.

Should androgenic anabolic steroids be considered in the treatment regime of selected chronic obstructive pulmonary disease patients?

            (Velema, Kwa et al. 2012) Download

PURPOSE OF REVIEW: Chronic obstructive pulmonary disease (COPD) is a widespread disease with high morbidity rates. Advanced stages can be complicated by unintentional weight loss and muscle wasting, which may contribute to increased morbidity and mortality. Reversal of weight loss increases muscle strength and exercise capacity and improves survival. This can partly be achieved by nutritional support, preferably combined with increase in exercise. Androgenic anabolic steroids (AASs), of which testosterone is the parent hormone, increase muscle size and strength. Due to these anabolic effects, AASs may emerge as a treatment option in COPD patients suffering from muscle wasting. RECENT FINDINGS: Seven trials investigated the effects of AAS in patients with COPD. Some studies also included nutritional therapy and/or a pulmonary rehabilitation program. Compared with placebo, AASs increase lean body mass (LBM) and muscle size. However, no consistent effects on muscle strength, exercise capacity, or pulmonary function are seen. SUMMARY: AASs increase LBM in patients with advanced stages of COPD. No consistent beneficial effect on other endpoints was demonstrated in the reviewed trials. However, probably higher doses of AASs are needed to exert a clinically meaningful effect on muscle strength or exercise capacity. Currently, no evidence is available to recommend AASs to all patients with COPD. In individual cases, treatment with AASs can be considered, particularly in men with advanced COPD, moderate-to-severe functional impairment, muscle wasting and on chronic corticosteroid therapy. Treatment with AASs should preferably be combined with a rehabilitation program and nutritional support. AASs should not be used in women or in men with symptomatic heart disease. When treatment with AASs is considered, intramuscular nandrolone-decanoate is preferred in a dose of 50-200 mg per week for a period of 12 weeks. However, the efficacy of AAS treatment in COPD patients needs further clarification in well designed, adequately powered clinical studies.


Bayliss, D. A. and D. E. Millhorn (1992). "Central neural mechanisms of progesterone action: application to the respiratory system." J Appl Physiol 73(2): 393-404.

Dimitropoulou, C., F. Drakopanagiotakis, et al. (2009). "Estrogen replacement therapy prevents airway dysfunction in a murine model of allergen-induced asthma." Lung 187(2): 116-27.

Hamidi, S. A., K. G. Dickman, et al. (2011). "17beta-estradiol protects the lung against acute injury: possible mediation by vasoactive intestinal polypeptide." Endocrinology 152(12): 4729-37.

Kamischke, A., D. E. Kemper, et al. (1998). "Testosterone levels in men with chronic obstructive pulmonary disease with or without glucocorticoid therapy." Eur Respir J 11(1): 41-5.

Manber, R., T. F. Kuo, et al. (2003). "The effects of hormone replacement therapy on sleep-disordered breathing in postmenopausal women: a pilot study." Sleep 26(2): 163-8.

Massaro, D. and G. D. Massaro (2004). "Estrogen regulates pulmonary alveolar formation, loss, and regeneration in mice." Am J Physiol Lung Cell Mol Physiol 287(6): L1154-9.

Saaresranta, T., P. Polo-Kantola, et al. (2006). "Menopausal estrogen therapy predicts better nocturnal oxyhemoglobin saturation." Maturitas 55(3): 255-63.

Sharma, S., A. Arneja, et al. (2008). "Anabolic steroids in COPD: a review and preliminary results of a randomized trial." Chron Respir Dis 5(3): 169-76.

Smith, A. M., R. T. Bennett, et al. (2008). "Characterization of the vasodilatory action of testosterone in the human pulmonary circulation." Vasc Health Risk Manag 4(6): 1459-66.

Svartberg, J., U. Aasebo, et al. (2004). "Testosterone treatment improves body composition and sexual function in men with COPD, in a 6-month randomized controlled trial." Respir Med 98(9): 906-13.

Svartberg, J., H. Schirmer, et al. (2007). "Reduced pulmonary function is associated with lower levels of endogenous total and free testosterone. The Tromso study." Eur J Epidemiol 22(2): 107-12.

Tofovic, S. P. (2010). "Estrogens and development of pulmonary hypertension: interaction of estradiol metabolism and pulmonary vascular disease." J Cardiovasc Pharmacol 56(6): 696-708.

Van Vliet, M., M. A. Spruit, et al. (2005). "Hypogonadism, quadriceps weakness, and exercise intolerance in chronic obstructive pulmonary disease." Am J Respir Crit Care Med 172(9): 1105-11.

Velema, M. S., B. H. Kwa, et al. (2012). "Should androgenic anabolic steroids be considered in the treatment regime of selected chronic obstructive pulmonary disease patients?" Curr Opin Pulm Med 18(2): 118-24.