In a recent Randomized Controlled Trial (RCT), we showed a slight but significant increase in maximal exercise tolerance (Tlim) in patients with chronic obstructive pulmonary disease (COPD) supplemented with vitamin B₁₂ during physical training, but without effects on oxygen uptake (V’O₂) kinetics beyond training alone (Paulin et al., 2017). Individuals with COPD are at risk of vitamin B₁₂ deficiency (Solomon, 2016) and hyperhomocysteinemia (HyCy) (Seemungal et al., 2007; Fimognari et al., 2009). Accordingly, HyCy is linked to numerous cardiovascular alteration (Ganguly and Alam, 2015) including histological changes in the heart (Piquereau et al., 2017), impaired global and segmental cardiac contractility (Kaya et al., 2014), or increased N-terminal-pro-B-type natriuretic peptide (NT-proBNP) secretion (Herrmann et al., 2007; Guéant Rodriguez et al., 2013). This prepropeptide is synthesized and stored as a high molecular weight mass propeptide from both the atria and ventricles, and released mainly under pressure/volume overload of the cardiac chambers, after cleavage of the active form of BNP, inducing natriuresis and vasodilatation (Calzetta et al., 2016). However, NT-proBNP has a longer plasma half-life and attains larger concentrations, besides being described as a significant marker of prognosis in heart failure (HF) (Cipriano et al., 2014). Moreover, vitamin B₁₂ or folic acid supplementation reduced NT-proBNP levels after 2 months of supplementation in subjects with NT-proBNP > 40 pg/mL (Herrmann et al., 2007), mitigated HyCy-induced cardiac dysfunction (Jeremic et al., 2018), and proved to be protective for mitochondrial function and cardiac contractile properties in a murine model, with lessening of upregulated atrial brain natriuretic peptide (ANP) (Piquereau et al., 2017). In contrast, an experimental study was negative for cardiac morphological alterations during HyCy induction (Taban-Shomal et al., 2009).

It is recognized that physical training causes a reduction in cardiac natriuretic peptides in HF (Cipriano et al., 2014) as well as which, a beneficial interaction between physical training and vitamin B₁₂ or folate supplementation in reducing HyCy has been suggested (König et al., 2003; Tyagi and Joshua, 2014). Of note, there is evidence of increased secretion of BNP associated with increased pulmonary vascular resistance during acute exercise in COPD (Fujii et al., 1999); a mechanism which demonstrates potential for attenuation through physical training. Thus, this secondary analysis aims primarily to explore the interaction between vitamin B₁₂ supplementation, physical training, and resting/exercise levels for NT-proBNP in a stable population of COPD patients. In addition, we sought to analyze possible associations between acute changes in NT-proBNP levels during exercise with Tlim, delivered power (watts, w), and oxygen uptake kinetics (V’O₂ time constant) on an ergometer, in order to explore the determinants of these possible changes. The central hypothesis was that there would be attenuation of neurohormone alterations with supplementation alone or combined with physical training.

As this is a secondary study of an already published RCT, the entire methodology has been previously described in detail (Paulin et al., 2017). Similarly, ethical considerations and consent details are published and recorded in the Brasilian Clinical Trials Registry (ReBEC number RBR-55f97c/2014). Additional unpublished methods will be considered in this exploratory study.

Selected baseline characteristics are described in Table 1 and detailed data have been previously published (Paulin et al., 2017). The groups were relatively balanced, with a significant difference only for Tlim at baseline (p = 0.041, Table 1). Despite a similar baseline PaO₂, the groups as a whole (n = 32) presented significantly reduced hemoglobin saturation through peripheral oximetry post-exercise (p < 0.0001). After interaction analysis, the supplementation with vitamin B₁₂ significantly changed the time course of NT-proBNP level during treatment (p = 0.048, Table 2 and Figure 2A). In addition, the final analysis did not support a significant change in NT-proBNP levels owing to high-intensity constant work-rate exercise (three-way RM ANOVA analysis and one-way RM ANOVA analysis with p-value > 0.05 for both, Table 2). A statistically significant correlation was observed only between V’O₂ time constant and ΔNT-proBNP level (Tlim – rest) at baseline (p = 0.049, Figure 2B) and 2 months later (p = 0.015, Figure 2C), considering all subjects (n = 32), with an absence of significant correlations between ΔNT-proBNP level and Tlim or delivered Power on the cycle ergometer (p > 0.05 for both). The pre- and post-training slopes representing the correlation between the V’O₂ time constant and ΔNT-proBNP (Figures 2B,C) were not significantly different for the groups as a whole (p = 0.259).

This exploratory study suggests that B₁₂ measurements and/or supplementation should be considered in future studies on cardiovascular morbidity in COPD subjects. This is in line with the overall pathophysiology of cardiovascular morbidity in COPD which has not yet been totally unraveled. In addition, we did not detect acute changes in plasma levels for NT-proBNP during high-intensity constant work-rate exercise. However, during the rest-to-exercise transition, patients with slower V’O₂ adjustments toward a steady-state appeared to secrete higher NT-proBNP levels.

Normal levels of vitamin B₁₂ do not rule out the possibility of cobalamin deficiency, and low-normal levels (200–300 pg/mL), when associated with risk factors for increased oxidative stress, demonstrate metabolic evidence of deficiency, with high levels of HyCy or methylmalonic acid (MMA), since this vitamin is inactivated by oxidation (Solomon, 2016). Our study population contained at least three subjects in each group with levels < 300 pg/mL of vitamin B₁₂ and supplementation significantly increased their levels after 8 weeks for the REHA+B₁₂ group and not significantly for the B₁₂ group (Paulin et al., 2017).

Several previously cited animal and human studies have described a relationship between vitamin B₁₂ or folic acid level, HyCy, and NT-proBNP levels. Although preliminary, our study points to an attenuating effect on NT-proBNP secretion with vitamin B12 supplementation, in agreement with previous studies (Herrmann et al., 2007; Guéant Rodriguez et al., 2013), despite weak evidence. In monitoring the evolution of HF, attenuated or stable levels of NT-proBNP secretion were associated with fewer total cardiovascular events (Franke et al., 2011). Although the mechanisms underpinning this small but significant effect are largely unexplored, downregulation of natriuretic peptide production by reducing homocysteine and MMA accumulation under certain conditions is possible (Piquereau et al., 2017).

Two important secondary findings were described. Although we did not find an acute change in NT-proBNP owing to acute exercise during high-intensity CWT, there was a small but significant relationship between an acute change in this neurohormone (Δ) and oxygen uptake kinetics. Our study differs from a previous study, which showed increased secretion of BNP after exercise in a population similar to ours during CWT (Fujii et al., 1999). These contradictory results occurred despite both presenting significant hemoglobin desaturation during exercise. Arterial hypoxemia is a known trigger for natriuretic peptide secretion (Fujii et al., 1999). Surprisingly, our study agrees with another study for NT-proBNP also under CWT in mild-to-moderate COPD (Wang et al., 2011). Differences may reflect other factors, such as the secretion-to-metabolization ratio of natriuretic peptides and differences in exercise protocol. Our data suggest that much of the secretion of natriuretic peptides during CWT occurs at the time of initial adjustment during rest-to-exercise transitions, where the sudden change in the cardiac chamber pressure/volume condition is additionally stressed by the direct effect of transmural pressure, due to the abrupt increase in intrathoracic respiratory pressure swings, following the kinetics of the increase in pulmonary arterial pressure during the first minute and posterior decay during exercise (Lonsdorfer-Wolf et al., 2004). In this sense, our study is in agreement with a previous study that showed a direct relationship between BNP secretion and oxygen deficit during constant work-rate exercise in HF (Brunner-La Rocca et al., 1999).

As a limitation of this study, we cite the small number of individuals, notwithstanding this being partially tempered by the strong study design. In addition, we did not measure homocysteine and MMA levels. Four subjects were not evaluated by echocardiography; however, clinical data, NT-proBNP levels, and CPET analysis were not compatible with major heart disease.

We suggest that vitamin B₁₂ supplementation could modulate NT-proBNP secretion, but the effects are small and further studies are needed. Moreover, we did not find an increase in neurohormone level caused by acute exercise per se; however, there was an association with slower V’O₂ adjustment during the rest-to-exercise transition. The significance of this neurohormone dynamic warrants more detailed studies, considering different modalities of exercise training, e.g., high-intensity 1-min bouts, with unexplored cardiac neuro- hormone signaling and unknown cardiovascular consequences.

The datasets generated for this study are available on request to the corresponding author.

The studies involving human participants were reviewed and approved by the Human Research Ethics Committee, affiliated to Brazilian Clinical Trial Registry and Federal University of Mato Grosso Do Sul. The patients/participants provided their written informed consent to participate in this study.

PM contributed to study design, literature search, data collection, analysis of data, and manuscript preparation and review. FP contributed to literature search, data collection, analysis of data, and manuscript preparation and review. LG contributed to study design, literature search, data collection, analysis of data, and manuscript review. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.