1. Introduction
The autonomic nervous system (ANS) is in charge of a series of reflex reactions. The maintenance of a stable internal environment depends on the balance between the sympathetic and parasympathetic systems. Changes in the activity of ANS can occur temporarily, but the majority of chronic diseases are associated with an important dysregulation of ANS. Asthma, diabetes, and obesity, common chronic diseases, are associated with each other as comorbidity, but also linked to meta-inflammation where a myriad of manifestations of autonomic alterations has been identified [
1,
2]. Those diseases share common risk factors also related to lifestyle and the increase of adiposity. For example, the study of Popa et al. showed that 61.7% of the population studied consumed fast food at least three times a week, increasing the risk for obesity 1.9 times, moreover 52.3% performed less than 150 min/week of physical exercise increasing the risk for such a condition 1.8 times [
3]. Undoubtedly, an increase in adipose tissue leads to clinical obesity. This increment frequently results in the secretion of adipokines, which in turn causes low-grade systemic inflammation. However, there is a connection between inflammation and ANS that regulates the pro and anti-inflammatory response in a dependent manner. The reactivation of chronic inflammatory illnesses like asthma is related to ANS activation. Likewise, overproduction of interleukin 6 by endothelial cells is also linked to ANS [
4]. To evaluate the functionality of ANS several tests are available. Most of them require specialized equipment and trained staff to perform the evaluation. Parasympathetic responses decrease with increasing body mass index (BMI) [
5]. Recently, a considerable amount of attention has been paid to hematological parameters as an innovative tool to evaluate a variety of ASN alterations related to an acute coronary syndrome in the hospital setting. [
6]. The rate of recovery of heart rate (HR) within the first minute after cessation of exercise, termed heart rate recovery (HRR), is a simple, accepted measure of ANS dysfunction, reflecting parasympathetic reactivation. The 6-min walking test (6MWT) is a standard, widely available, clinical test of cardiopulmonary reserve of patients with heart failure and pulmonary diseases like pulmonary hypertension, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF). The 6MWT is a submaximal exercise test and a simpler alternative to the cardiopulmonary exercise test [
7,
8].
Different abnormal HRR (aHRR) cutoffs have been utilized in the past. For example, a cutoff of ≤42 beats/min after 2 min of recovery predicted cardiovascular death. A cutoff of ≤22 beats/min during the first 2 min after exercise anticipated the presence of coronary artery disease and death in males. Moreover, a decline of ≤42 beats/min after 2 min is associated with impaired fasting plasma glucose and an abnormal triglycerides/HDL cholesterol ratio (highest quartile) in healthy adults. Clinical deterioration in idiopathic pulmonary arterial hypertension is associated with less than 16 beats/min at the first minute. Abnormal HRR of 13 and 22 beats/min at the first and second minute, respectively, are good predictors of mortality in idiopathic pulmonary fibrosis. In chronic obstructive pulmonary disease (COPD), a cutoff of ≤12 beats/min in the first minute is related to pulmonary function, oxygen saturation, and disease severity [
9].
HRR is a predictive biomarker of clinical worsening, hospitalization, and survival of different diseases such as connective tissue disease-associated pulmonary hypertension, idiopathic pulmonary fibrosis, poorly controlled severe asthma, cystic fibrosis, chronic obstructive pulmonary disease (COPD), bronchiectasis, heart failure, and diabetes [
10,
11,
12]. HRR correlates with nutritional state and biochemical parameters. Some studies stated that HRR values at first (HRR1) and second (HRR2) minute post-exercise did not differ substantially from each other and are a significant risk factor for death from any cause, cardiovascular death, and even non-cardiovascular death [
13,
14].
Acute coronary syndrome has been associated with changes in blood cell count [
14] and proposed as biomarkers of prognosis in those patients but there is not enough recent evidence regarding the relationship between cellular and biochemical measurements commonly used in clinical settings with HRR. The goal of this study was to evaluate the relationship between HRR and anthropometry, body composition, hematological and blood chemistry parameters, and whether these have possible use as autonomic dysfunction biomarkers.
2. Materials and Methods
We undertook this study at the Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas (INER) in Mexico City at 2240 m above sea level and treating mainly uninsured patients. The institutional Ethics Committee granted ethical approval, code C22-14, and all the participants signed an informed consent form. Sensitive information was removed from the collected data to preserve the anonymity of the participants. We conducted the study under the ethical principles of the Declaration of Helsinki.
We conducted a cross-sectional study of ambulatory patients with controlled asthma from the Asthma Clinic of the INER and patients with type 2 diabetes (T2D) recruited from the nearby Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ). Males and females aged 18 to 65 years, free of major diabetes-related chronic complications, and displaying HbA1c levels < 12%, and healthy volunteers, were invited to participate. Patients with a previous diagnosis of a cardiac congenital condition, autoimmune disease (including type 1 diabetes), oncologic disease, total cholesterol > 200 mg/dL, triglycerides > 300 mg/dL, thyroid disease, a body mass index less than 20 or more than 40 Kg/m2, individuals who had smoked at least 100 cigarettes and who smoked at the time of the study, treatment with beta-blockers and patients with an acute respiratory infection or asthma exacerbation within the previous 6 weeks at the time of the study were excluded. We did not analyze data from patients unable to perform the 6MWT.
All participants were asked to stop short- and long-acting inhaled beta2 agonists as well as anticholinergic drugs such as ipratropium bromide 12 h before the 6-min walking test (6MWT). If they could not stop their medication, the 6MWT test was not performed. Body composition (BIA) was estimated with a bioimpedance analyzer (Seca
® mBCA-514 analyzer, Hamburg, Germany). Anthropometric measurements including neck and waist circumference, complete blood count (CBC), and blood chemistry (BC) were obtained. Respiratory function was evaluated with forced spirometry (NDD Easy On-PC, ndd Medical Technologies, Inc., Massachusetts, USA) following the ATS/ERS 2005 guidelines; fractional exhaled nitric oxide (FeNO) and exhaled carbon monoxide measurements were also performed. Exhaled carbon monoxide was measured with a Smokerlyzer
® (Bedfont Scientific Ltd., Harrietsham, UK); FeNO measurements were conducted at a constant flow of 50 mL·s
−1 in line with the ATS/European Respiratory Society (ERS) recommendations using a portable handheld analyzer (NObreath
® analyzer; Bedfont Scientific Ltd., Harrietsham, UK). The 6MWT was performed indoors, along a flat, straight, 30 m walking course, according to the guidelines of ATS/ERS 2014 [
15]. HRR was calculated as the difference between peak heart rate and heart rate 1, and 2 min following test cessation; relative HRR (rHRR) was obtained with the following formula:
where
HRf is final heart rate and
HRx is heart rate at rest.
Arterial blood pressure and blood samples were obtained before the 6MWT and the lung function tests; all tests were carried out during fasting at the same hour of the day to avoid circadian variations in the autonomic nervous system activity. Arterial blood pressure was measured after a 5-min rest in a seated position by auscultation of the brachial artery with a stethoscope (3M™ Littmann
® Classic III™, Two Harbors, MN, USA) and an aneroid sphygmomanometer (767 Mobile Aneroid Welch Allyn, Inc., Skaneateles Falls, NY, USA) to detect the appearance and disappearance of the Korotkoff sounds and performed by the same device and same pneumologist specialized in respiratory tests to minimize inter-device and interobserver variability. Blood pressure was categorized according to the American Heart Association criteria [
16]. The laboratory analyses were performed at the institutional laboratory. The BC analysis included the following parameters: albumin, triglycerides, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), no-HDL, phospholipids, apolipoprotein A (Apo-A), apolipoprotein B (Apo-B), glucose, glycosylated hemoglobin (HbA1c), Insulin, C-reactive protein (CRP), urea, blood urea nitrogen (BUN), uric acid, and creatinine. From the BC analysis, we obtained the following indices: homeostatic model insulin resistance (HOMA-IR); Castelli risk index I (total cholesterol/high-density lipoprotein) and Castelli risk index II (low-density lipoprotein/high-density lipoprotein); atherogenic coefficient (AC; (TC-HDL)/HDL); Apo index; and atherogenic index of plasma (AIP; Log10 (TG/HDLc)). To evaluate the relationship between HRR and the measured parameters, the patients were reclassified into several categories as follows: sex, BMI, presence or absence of metabolic syndrome according to the National Cholesterol Education Program’s Adult Treatment Panel III/NCEP-ATPIII, and diagnosis of type 2 diabetes mellitus. Insulin resistance (IR) was determined through homeostatic model assessment (HOMA-IR) using a calculator (
https://amhigo.com/mi-diagnostico/calculadoras/indice-de-resistencia-a-la-insulina-homa-ir, accessed on 19 October 2019) with cutoffs for the Mexican population. We also obtained values for waist-to-height-ratio (WHtR) and neck circumference. Finally, based on the atherogenic indices, the groups were classified as low or high risk.
Statistical Analysis
The baseline characteristics of the studied population were stratified as follows: abnormal HRR at minutes 1 (aHRR1) and 2 (aHRR2) with cutoff values set at ≤8 and ≤11 bpm, respectively, based on the first quartile of all the data; presence or absence of asthma; type 2 diabetes and metabolic syndrome (MetS); BMI classification; WHtR; and high or low atherogenic risk.
All data are expressed as the median followed by the interquartile range 25–75 (IQR). Correlations between variables were performed with a Spearman’s test. Comparisons between more than two groups were made using a non-parametric Kruskal–Wallis test followed by a post-hoc Dunn’s test. A Wilcoxon rank-sum test was used to compare two groups. A chi-square test was used to compare frequencies between two categorical variables. All statistical analyses were conducted in STATA 13 (StataCorp. 2013. Stata Statistical Software: Release 13. College Station, TX, USA: StataCorp LP). Graphs were created using GraphPad Prism version 6.00 for Windows (GraphPad Software, La Jolla, CA, USA).
4. Discussion
The 6MWT is a clinically validated submaximal exercise test [
8], similar to activities undertaken in everyday life with relevant association with patients’ symptoms, quality of life, all-cause mortality, hospital readmission, and the combined endpoint of death or read-mission in patients with heart failure [
19]. Post-exercise HR and HRR evaluate vagal nerve integrity and measure cardiac autonomic activity.
BIA is a non-invasive assessment of tissues, based on electrical impedance, and affected by changes in health status (e.g., nutritional state, swelling, infections, and disease). Our study proposes a relationship between BIA measurements and the HR recovery after exercise, implying a dysautonomic state of the patients. Phase angle, intracellular water, and hydration percentage showed statistical differences related to dysautonomia as determined by a slow HRR. At present, there are different indices to establish nutritional condition (e.g., BMI, neck and waist circumference, waist-to-height ratio); however, BIA provides additional information regarding the nutritional state than other parameters. Phase angle showed a significant correlation with HR, HRR, and rHRR whereas other indicators of a nutritional condition such as BMI, WHtr, NC and WC, did not show any statistical association.
The SMM parameter showed marginal and discrete statistical differences. This result suggests an increase in muscle mass, possibly cardiac muscle mass. Bioimpedance cannot distinguish between skeletal and visceral muscle mass. Disturbances of the autonomic nervous system are present in heart hypertrophy of different etiologies, such as hypertension or atherogenic risk, and heart failure to compensate for the cardiac output [
20].
Studies report that the immune response can contribute to autonomic nervous system dysfunction through immune signaling molecules like cytokines, affecting heart rate variability [
21], which correlates with HRR [
22] through the depression of action potential [
23]. The HRR results presented in this study suggest the presence of integrated immune-neuroendocrinal interactions, where white blood cell counts contribute to aHRR values. HRR depends on a physiological regulation mechanism. The sympathetic nervous system regulates the immunological cells, immune organs, and the responses of acute phase reactions [
24]. The observed white blood cell count values suggest a relationship between circulatory inflammatory cells and changes in HRR1, rHRR1, and aHRR1. Even though the cell counts found in both HRR and aHRR subjects were within the normal reference limits, the differences between groups had statistical significance. A cross-sectional study displayed similar results; there was an increase in white blood cell count and PCR in aHRR1 subjects. This phenomenon was independent of disease status, blood pressure, blood lipids, body size, smoking, and fasting blood glucose [
20].
Hematological parameters are related to oxygen transport and iron deficiency. Total erythrocytes, hemoglobin, and hematocrit showed a correlation and discrete changes related to HR recovery.
Insulin resistance syndrome contributes to HRR in adolescents, adults, and elderly men [
25,
26]. It correlated with malnutrition and a high frequency of metabolic syndrome as well. The increase in serum insulin is related to chronic inflammation, and both stimuli contribute to the overstimulation of the sympathetic nervous system. Additionally, several prospective studies indicate that impaired glucose tolerance at baseline is an independent predictor of cardiovascular disease, even among the nondiabetic population. Moreover, metainflammation is related to the development of atherosclerosis [
21,
27]. Our results are in accordance with the prevalence of dysautonomia in patients with CVD risk, which was observed in many of the patients in HRR during the first minute of rest after the maximal and submaximal exercise test [
21]. Furthermore, we observed that this effect persisted within the first three minutes after the 6MWT suggesting that even when comorbidities such as MetS, diabetes, and asthma are under control, patients have a high cardiovascular risk that increases susceptibility to atherogenic processes. We observed that HR at 1 and 2 min after the 6MWT had a better correlation against the calculated HRR and rHRR. Therefore, HR should be considered together with HRR parameters.
In summary, this study describes respiratory-related parameters, body composition, and cellular and biochemical blood changes in a population with asthma, type 2 diabetes and obesity, under medical control. These medical conditions lower life expectancy due to a higher risk of developing fatal cardiovascular events. Furthermore, overweight and obesity per se are also significant risks for CVD and have a negative impact on fitness performance independent of age [
21]. The 6MWT test should be included in the battery test in hospitalized and ambulatory patients as part of the evaluation of illness progression and treatment response.
The time difference in heart rate recovery after cessation of exercise is a biomarker of the outcome. Van de Vegte, et al. [
22] reported an association of change in heart rate between 10 s and 1 min after exercise cessation and found that 10 s is a better predictor of mortality for all-cause and coronary artery disease mortality. The evaluation at 1 or 2 min after exercise cessation is used in clinical and sports assessment. Lamberts et al. [
23] reported that HRR helps to monitor changes in endurance performance and contributes to a more accurate prescription of training load in well-trained and elite cyclists. High HRR in athletes is related to cardiovascular fitness; however, research in athletes is limited; moreover, the methodologies to evaluate HRR used in these studies are varied [
28]. On the other hand, clinical HRR has been used as a predicted biomarker for worsening and deadly cardiorespiratory and metabolic diseases. Few studies aim to explain the cellular and biochemical parameters involved in nervous dysautonomia.
Our study suggests that HR, HRR, and rHRR are related to biochemical, cellular, and physiological responses. Patients with dysautonomia showed lower oxygen saturation that harms the microvasculature because of oxygen restriction. These physiological changes contribute to the disruption of the autonomous nervous system. They also promote subclinical immunological and hematological modifications to compensate for a hypoxic environment. Immune cells promote inflammation that contributes to insulin resistance syndrome concomitant with dysregulation of the hydric state, expressed by water body composition. Inflammation and insulin resistance favor autonomic nervous dysregulation and the integrity of the vagal nerve.
Heart rate variability (HRV) is another clinical tool used to evaluate autonomic nervous dysregulation. One methodological advantage of HRR using the 6MWT over HRV is the procedure and the cost of the equipment, which is more expensive for HRV compared to the 6MWT; however, HRV statistical or domain parameters are more refined than those of resting HR or the estimation of HRR. Nevertheless, the use of HRR methodology results in a low-cost test that is easy to perform and quickly interpreted by health or sports staff. Even though both HRV and HRR are accepted as non-invasive measurements of autonomic dysfunction, and useful biomarkers for worsening and death prognosis, there is still a debate about the lack of correlation between HRR and HRV, influenced by independent aspects of cardiac function (respiratory frequency, temperature, noise) [
29]. These two types of measurement provide self-sufficient and complementary information on cardiac parasympathetic function. Further studies are needed to establish a better correlation and determine how biochemical and hematological biometry explain or contribute to autonomic dysfunction.
Limitations of the present study are the low number of patients recruited and the poor relation of the biochemical and hematological parameters to the extended reported outcomes related to HRR, such as survival and clinical worsening. However, a relevant contribution of the present study is the relation of HRR and biochemical, cellular, and physiological parameters that can explain autonomic dysfunction and the increase of atherogenic risk to this condition.