Background
The peripheral arterial chemoreceptors are key O
2-sensors for O
2-homeostasis in normoxia and hypoxia during all phases of life [
1‐
4]. Upon hypoxic stimulation, the chemoreceptors trigger a reflexogenic hypoxic ventilatory response (HVR) which along with neurohumoral responses contributes a great portion to resting minute ventilation limiting arterial O
2-desaturation e.g. during sleep [
1,
2,
5,
6]. The isocapnic HVR, as a measure of carotid O
2-chemosensitivity, is considered to be a hereditary and therefore relatively stable individual feature and at the same time reveals a surprisingly large interindividual variability [
7,
8], which determines and predicts intolerance of healthy subjects to severe hypoxia, e.g. at high altitude [
9,
10], and fatal respiratory failure in rare cases of genetically abolished HVR [
2]. A low HVR may especially become critical with severely hypoxemic clinical conditions like chronic obstructive pulmonary disease (COPD) or sometimes obstructive sleep apnea [
11,
12] which, however, involves a long-term potentiation of HVR with a rather complex pathophysiological role [
3,
4,
13]. In healthy adults, a limited number of factors beside drugs reportedly lead to acquired modifications of HVR including acute and chronic hypoxic exposure, aging and, potentially, endurance training [
3,
14‐
17].
A critical HVR attenuation has, however, been extensively discussed to result from long-term intrauterine and/or early postnatal nicotine exposure, thereby possibly linking the sudden infant death syndrome to maternal smoking in a dose–response-fashion [
4,
18‐
21]. Among possible mechanisms suggested for such nicotine-induced impairment of chemoreceptor O
2-sensing were alterations within the β
2-subunit of the nicotinic acetylcholine receptor because the nicotine effect was abrogated or mimicked in related β
2-subunit mutants [
22,
23]. In humans, evidence for an impaired chemoreceptor O
2-sensing through nicotine exposure appears to be preliminary and restricted to studies in infants of smoking mothers in the context of sudden infant death: Healthy, ≤3 months old, term or preterm infants exposed to maternal cigarette smoking/nicotine revealed a weakened (poikilocapnic) HVR and awakening response [
18,
24,
25].
However, the important question, whether in adulthood long-term smoking may affect HVR, has remained surprisingly understudied. One earlier study by Kawakami et al. [
26] in smokers (SM) and their non-smoking (NSM) homozygote twins failed to demonstrate a smoking-related HVR attenuation after a 3-h-abstinence which is insufficient to eliminate nicotine with an in-vivo half-life of 2 h, as already speculated by these authors themselves. In fact, subsequent studies, including one from the same group, have shown an acute HVR-increase through smoking in both, SM and NSM probably mediated through carotid chemoreceptors [
27‐
30] which might have masked a possible HVR attenuation in the elegant study by Kawakami et al. [
26] in twins.
The present study intended to clarify whether or not healthy adult SM reveal a substantial reduction of their isocapnic HVR compared to NSM, when abstaining long enough (12 h overnight) from smoking to eliminate nicotine. According to a representative diurnal profile of smokers, plasma nicotine levels accumulate (to between 10 and 30 ng/ml) in the evening and are eliminated to below 10% within 10 h in healthy adults, while cotinine as a major metabolite is eliminated by about 50% [
31,
32]. Furthermore, we evaluated the possible acute masking effect of subsequent re-exposure to cigarette smoke. Because smoking may acutely and chronically induce oxidative stress [
33,
34], we also assessed the thiol/disulphide redox state in the plasma and in peripheral blood mononuclear cells (PBMCs), which both may massively affect HVR [
35,
36]. In addition, we matched SM and NSM for factors known to affect HVR, like age, sex, and BMI and excluded differences in plasma levels of glucose, HbA1c or potassium [
15,
16,
37,
38]. We found a significant, large reduction of isocapnic HVR in healthy male adult SM compared to NSM, which was virtually completely masked by acute enhancement through smoking a single cigarette.
Results
The anthropometric data of age-matched healthy NSM and SM reflected a normal nutritional status and arterial blood pressure values with no significant differences between the two groups (Table
1). The routine parameters of pulmonary function were comparable between both groups and excluded respiratory diseases of relevance for HVR assessment such as bronchial asthma. Mainly due to a lower, albeit non-significant, respiratory frequency (about −20%,
P = 0.078), resting minute ventilation was found to be significantly lower in SM (about −13%,
P = 0.006) compared to NSM. However, this was not associated with any difference in PetCO
2 levels between the two groups because, at similar VO
2, SM had almost significantly lower VCO
2 (about −8%,
P = 0.058) compared to NSM, i.e., SM tended to have a lower respiratory quotient (RQ,
P = 0.088). Expectedly, no difference in peripheral arterial O
2-saturation at rest was detected between SM and NSM.
As a main present finding, isocapnic HVR in terms of both, absolute and normalized values (for individual BMI) showed a highly significant reduction (about −35%) in SM compared to NSM (Fig.
1, Table
1). PetCO
2HVR during HVR measurement was well kept at isocapnic levels i.e. at prevailing individual resting normoxic values and, importantly, was virtually identical between SM and NSM (Table
1).
Among the traditional vascular risk factors (Table
2) of these two adult groups, plasma lipids including total cholesterol, VLDL, LDL, HDL, and triglycerides as well as systolic and diastolic arterial blood pressure, fasting glucose, HbA1c and homocysteine were all found to be within the normal range with slightly, though significantly, higher levels observed for triglycerides, total cholesterol, and VLDL in SM compared to NSM. Notably, SM had a considerable and significantly higher level of oxLDL (about 68%) compared to NSM. In contrast, the plasma cysteine and cystine as well as the intracellular GSH and GSSG showed no significant smoking-related differences. While SM showed significantly higher plasma levels of circulating ICAM-1 (about +41%) as another non-traditional cardiovascular risk factor, no significant differences were found for VCAM-1 and TNFα levels compared to NSM.
Table 2
Blood cardiovascular risk factors in non-smokers (NSM) and smokers (SM): plasma lipids, oxidized LDL, basal glucose, extra- and intracellular thiol redox state, adhesion molecules and TNF-α
Triglycerides | (mg 100 ml−1) | 66.4 ± 5.7 | 94.0 ± 7.5 | 0.005c |
Total cholesterol | (mg 100 ml−1) | 178 ± 7 | 206 ± 8 | 0.016b |
VLDL | (mg 100 ml−1) | 14.3 ± 1.2 | 23.1 ± 3.1 | 0.012b |
LDL | (mg 100 ml−1) | 121 ± 7 | 140 ± 9 | 0.094 |
oxLDL | (U l−1) | 52.9 ± 5.4 | 88.6 ± 13.6 | 0.021b |
HDL | (mg 100 ml−1) | 44.3 ± 1.8 | 45.3 ± 2.6 | 0.734 |
Glucose | (mg 100 ml−1) | 79.7 ± 2.8 | 73.5 ± 2.7 | 0.115 |
HbA1c | (%) | 5.30 ± 0.07 | 5.25 ± 0.06 | 0.590 |
Homocysteine | (μM) | 9.0 ± 0.5 | 9.2 ± 0.5 | 0.715 |
Cysteine | (μM) | 7.66 ± 0.32 | 7.47 ± 0.35 | 0.687 |
Cystine | (μM) | 40.8 ± 1.1 | 43.3 ± 1.2 | 0.126 |
GSHintracellular | (nmol mg−1) | 15.3 ± 2.2 | 18.3 ± 1.8 | 0.303 |
GSSGintracellular | (nmol mg−1) | 2.94 ± 0.42 | 2.37 ± 0.50 | 0.397 |
ICAM-1 | (ng ml−1) | 378 ± 30 | 533 ± 35 | 0.002c |
VCAM-1 | (ng ml−1) | 791 ± 47 | 726 ± 40 | 0.297 |
TNF-α | (pg ml−1) | 24.3 ± 4.8 | 22.4 ± 2.0 | 0.717 |
In a subgroup of SM (
n = 10) we furthermore repeated HVR measurement immediately after smoking one cigarette (Fig.
2). This re-exposure led to a highly significant acute increase in HVR (
P = 0.005) as compared to the condition of 12-h-abstinence from cigarettes. The mean increase in HVR evaluated in SM amounted up to 30% (at a rather wide inter-individual variability), thus reaching a level that was not significantly different from that of NSM (without experimental exposure to cigarette smoke).
According to explorative correlation analysis, the number of ‘pack years’ (range: 6–60) were neither significantly related to the individual HVR (during abstinence from cigarettes or upon re-exposure to one cigarette, with or without normalization for BMI) nor to any other ventilatory parameter given in Table
1. This was also true when controlling for the factor age in a multivariate regression approach. However, a positive correlation of ‘pack years’ was found to SM’s oxLDL (
r = 0.421,
P = 0.057) and HbA1
C (
r = 0.475,
P = 0.022) while a negative correlation existed to intracellular GSSG (
r = −0.474,
P = 0.026). The number of daily smoked cigarettes (range: 15–50) showed no significant relation to any measured parameter.
Discussion
To the best of our knowledge this cross-sectional study is the first to detect a significant and substantial reduction of HVR in healthy, adult long-term SM under conditions of 12-h of abstinence from cigarettes. In addition we demonstrate - well in line with previous findings by others - that upon re-exposure to cigarette smoke HVR is acutely increased to a level that is virtually indistinguishable from that of NSM. This may lead to the important conclusion that a chronic HVR attenuation in SM is obviously masked during daytime smoking habit and therefore may have been overlooked in previous studies with insufficient nicotine abstinence.
The difference in HVR between SM and NSM was demonstrated with a power of 0.97 (
p ≤ 0.05) at a normal distribution in both groups and a large HVR overlap at the expected wide variability within both the SM and NSM sample (Fig.
1). Unlike the hypercapnic ventilatory response, the isocapnic HVR as a measure of peripheral carotid chemoreceptor O
2-sensitivity is considered as a quite stable, partly hereditary, individual feature with a uniquely wide inter-subject variability [
2,
3,
7,
8] which - in line with a major chemoreceptor contribution to resting ventilator drives [
2‐
5] - is considered to determine (in-) tolerance to high altitude and hypoxemia with pulmonary diseases [
1,
6,
9,
10,
12,
13]. Interestingly, smoking may aggravate the physiological O
2-desaturation during sleep [
39]. A low HVR in SM may therefore possibly represent an understudied, novel link between smoking and the risk for aggravated O
2-desaturation and play a potential role within the complex pathophysiology of COPD or of weaning from artificial respiration. Our finding may therefore warrant more detailed human studies addressing effects of smoking duration, intensity and cessation, possible interaction of gender, aging and additional cardiovascular risk factors, especially those associated with altered HVR like hypertension and obesity [
37].
Measurement of ventilator drives in humans is well-known to be easily confounded by several factors, many of which were carefully considered in this study. 1) Isocapnia during HVR was well controlled, i.e. the PetCO
2 was kept at the level observed during normoxic baseline and was virtually identical between SM and NSM (Table
1). 2) Plasma potassium levels which affect peripheral chemoreceptors were not significantly different between SM and NSM (4.07 ± 0.06 vs 4.32 ± 0.12 mM). 3) We furthermore showed, that the plasma thiol (cysteine and homocysteine) and cystine as well as the intracellular levels of GSH and GSSG were comparable between SM and NSM (Table
2). This is important as interventional studies by us and others have demonstrated a large HVR increase with acute supplementation of thiol-compounds beside a significant correlation between HVR and the intracellular GSH [
35,
36]. Though a smoking-related difference in the thiol redox state has previously been reported [
33,
34], the present study conducted in a strictly postabsorptive and smoking-abstinent state demonstrated good comparability of the redox state between SM and NSM. Furthermore, SM and NSM had similar whole blood levels of homocysteine, a thiol compound that clearly interacts with other protein- (albumin-) bound thiols like cysteine by disulphide exchange [
40]. 4) Another factor influencing HVR is plasma glucose, which was shown to be sensed along with pO
2 by peripheral chemoreceptor type 1 cells, such, that hypoglycemia massively increases the HVR in humans [
38,
41]. The present data were obtained at comparable, fasted blood glucose and HbA1c levels with a tendency towards lower glucose levels in SM, which would rather increase than decreases HVR (Table
2).
Possible mechanisms behind the observed HVR attenuation within the chemoreceptors in adult SM remain speculative at present and may include (epigenetically) altered expression of hypoxia-inducible factor 1α and/or 2α [
3], an alteration of the β
2-nicotinic acetylcholine receptor subunit in the chemoreceptors (or brainstem centers) as a possible target of nicotine [
22,
23], dopamine-mediated alterations following an upregulation of the tyrosine hydroxylase within the carotid body as shown in developing rats after nicotine exposure [
42], or other factors. Interestingly, HVR in SM at both conditions tested (i.e. during abstinence or upon re-exposure to a single cigarette) was unrelated to pack years (range: 6–60 pack years) or daily smoked cigarettes (range: 15–50), even when controlling for the factor age (range: 22–53 years), which appears to exclude a simple dose-dependent mechanism.
At present, we cannot strictly exclude a rather speculative effect of slight elevations of plasma lipids, oxLDL, or ICAM-1 in SM compared to NSM, because these factors may be associated with endothelial dysfunction, which may not spare out the carotid body arteries. However, HVR was unrelated to these risk factors and considerably higher lipid levels have previously been demonstrated not to affect HVR [
43].
As a limitation, this study includes no data on nicotine or cotinine plasma levels to quantify overnight nicotine elimination, i.e. compliance to abstinence from cigarettes or to demonstrate the nicotine increases upon re-exposure to cigarettes. However, our study demonstrates virtually identical plasma thiol (cysteine) levels between SM and NSM on arrival at our laboratory at 8:00 a.m. This may exclude smoking within 1 h prior to blood sampling, because plasma thiol (cysteine) decreases by >50% upon smoking of a single cigarette and takes one hour to return to pre-smoking level [
34]. Given that no cigarette was smoked on test day after an 8-h-sleep between 7:00 (reminding phone call) and 8:00 a.m. and that subjects were under observation at the laboratory thereafter until completion of HVR between 9:00 and 10:00 a.m., a 10-h-abstinence from cigarettes can be assumed. The ‘last’ cigarette was reported by phone call or SMS before 11:00 p.m. on the evening before which would yield a 12 h abstinence. In addition, beside the thiol plasma level, our data on the intracellular thiol redox state show similar levels between SM and NSM.
Even with excellent compliance we cannot presently exclude confounding effect of the nicotine metabolites like cotinine (with an in-vivo half-life of around 20 h) and, furthermore, of carboxyhemoglobin (CO-Hb) not detected by the peripheral O
2-saturation measurement. Because the nicotine clearance depends on various factors including age, gender, hepatic function and blood flow (with large postprandial increase), renal function and factors within the smoking habit itself, further detailed studies on the present observation appear warranted [
31,
32]. Thereby, beside the individual smoking history the early childhood cigarette smoke or intrauterine nicotine exposure may have to be assessed as well, to identify relevant factors in smoking-related HVR alterations (chronic reduction as opposed to acute enhancement).
Furthermore, due to a lack of studies in humans, we can only speculate on the finding of an almost significantly lower VCO
2 and RQ (at similar VO
2) in SM compared to NSM, which obviously yielded similar PetCO
2 at significantly lower V
E in SM. A previous study in rats has described a (sub-) acute lowering of RQ through nicotine at unchanged resting energy expenditure [
44]. Whether this effect is relevant to humans and (still) present (or reversed) upon the presently studied short-term nicotine abstinence, remains unclear at present.
Importantly, the present study at the same time confirmed an acute HVR increase upon (re-) exposure of SM to cigarette smoke to an extent that was sufficient to completely mask the chronic HVR attenuation discussed above (Fig.
2). In fact, the earlier study by Kawakami et al. [
26] comparing monozygotic twin SM and NSM, unfortunately, failed to detect differences in HVR, likely because the only 3-h-abstinence from cigarettes used in that study was insufficient to eliminate acute stimulatory affects. Such acute HVR enhancement was, however, subsequently shown, in both SM and NSM as well as for mammals, including one study from the same group [
27‐
30].