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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 30, No 2, March/April 2019
AFRICA
109
Official permission for the study was obtained by the ethics
committee of the Medical University of Plovdiv with a protocol
reference number P-1548/13.03.2014. Guidelines for proper
human care were fully implemented, following the Helskinki
agreement and human rights. The subjects received all relevant
information about the study, including its aim, protocol, tests
and any potential side effects. A signed informed consent was
obtained from all subjects prior to inclusion in the study and a
questionnaire about their physical status was completed.
During the experimental session, the participants did not
take any medication, coffee, alcohol or tobacco for at least two
hours prior to the test. A physical examination was carried out,
including an electrocardiogram (ECG), which was reviewed by
a cardiologist to exclude cardiovascular abnormalities or any
rhythm or conductive disorders. No side effects or symptoms
were reported during the session.
The subjects were placed in a supine position on a comfortable
bed, in a quiet, well-aerated room with constant light and
ambient temperature, in the absence of any distracting factors.
They were instructed to stay calm, without excessive voluntary
movements or speaking and to breathe normally. During the
first 10 minutes of the visit, the participants breathed ambient
air at an altitude of 130 m (Plovdiv, Bulgaria). Subsequently, air
with an oxygen concentration of 12.3
±
1.5%, corresponding to
an altitude of about 4 200 m, was administered for one hour via
a full-face mask using a hypoxicator (AltiPro 8850 Summit+,
Altitude Tech, Canada). The protocol did not include any
changes in the barometric pressure (normobaric hypoxia).
At the end of the hypoxic session, the mask was removed
and the subjects were exposed to ambient air. Four-channel
ECG (H3+, Mortara Instruments, Milwaukee, USA) and pulse
oximetry (CMS50F, Contec Medical Systems, Qinhuangdao,
China) were recorded during the whole time. The ECG recordings
were reviewed and R-R intervals were extracted automatically by
H-Scribe 5 software (Mortara Instruments, Milwaukee, USA).
Five-minute samples were selected from the end of the
pre-hypoxic and hypoxic periods, and immediately after hypoxic
exposure for the subsequent analysis. After removing trends, data
were analysed using Kubios HRV software and domain, and
frequency domain and non-linear parameters were calculated.
13
Fast Fourier transform was used for calculation of the frequency
domain parameters using Welch’s periodogram with a window
length of 256 seconds and 50% overlap.
The following parameters were derived from the R-R data:
total power (TP) and standard deviation of the intervals between
normal beats (SDNN) as measures of overall autonomic
regulation; absolute power and normalised units (nu) of high-
frequency (0.15–0.40 Hz) and low-frequency (0.04–0.15 Hz)
spectral components, respectively, reflecting parasympathetic
nervous system (PNS) activity and combined sympathetic (SNS)
and PNS activities. The ratio LF/HF was also calculated as an
index of sympatho-vagal balance. The mean square root of
successive R-R interval differences (RMSSD) is a time domain
parameter associated with parasympathetic activity.
In addition to the linear methods described, three commonly
used non-linear parameters were applied. These included
standard deviations SD1 and SD2 of the Poincaré plot – SD1
related to fast beat-to-beat variability in data and SD2 describing
longer-term variability of R-R.
14,15
Sample entropy (SampEn)
was also collated as an index for the overall complexity and
predictability of HRV time series.
16
Statistical analysis was performed with the independent
samples
t
-test (SPSS v.17.0) when comparing the group of
smokers and non-smokers, and paired samples
t
-test was used
to compare intra-group changes between hypoxic and post-
hypoxic periods. Normality of distribution was assessed with the
Shapiro–Wilk test. Skewness of distribution of some parameters
(absolute spectral powers) was normalised by means of natural
logarithmic transformation.
Results
There was no difference in peripheral capillary oxygen saturation
(SpO
2
) between smokers and non-smokers under normoxic
Table 1. Comparison of age and main anthropometric
parameters between the two groups
Parameters
Non-smokers
Smokers
p
-value
Age (years)
28.0
±
7.4
28.1
±
4.3
0.949
Height (cm)
179.9
±
6.2
179.7
±
4.3
0.941
Weight (kg)
78.3
±
6.6
77.4
±
10.0
0.762
Body mass index (kg/m
2
)
24.3
±
2.4
24.0
±
3.2
0.796
Table 2. Comparison of oxygen saturation, heart rate and HRV parameters between ‘healthy’ smokers and
non-smokers before, during and after exogenous hypoxia provocation
Before hypoxia
Hypoxia
After hypoxia
Parameters
Non-smokers
Smokers
p
-value Non-smokers
Smokers
p
-value Non-smokers
Smokers
p
-value
SpO
2
(%)
96.8
±
1.2
96.3
±
1.6
0.276 88.2
±
5.9
91.4
±
1.4
0.026
96.8
±
1.1
95.9
±
1.1
0.021
HR (beat/min)
62.2
±
9.0
79.3
±
14.2
0.008 65.1
±
11.1
77.3
±
13.8 0.007
61.9
±
9.0
74.5
±
13.5 0.006
SDNN (ms)
62.0
±
32.1
40.3
±
16.2
0.013 75.7
±
34.8
57.9
±
18.3 0.167
87.8
±
39.2
61.9
±
9.0
0.021
RMSSD (ms)
59.3
±
32.8
24.9
±
12.5 < 0.001 65.9
±
40.1
34.3
±
17.1 0.003
75.1
±
45.9
34.9
±
20.0 0.001
lnTP (ms
2
)
7.7
±
1.1
7.0
±
0.8
0.046
8.2
±
1.7
7.9
±
1.0
0.309
8.5
±
0.9
7.9
±
0.9
0.068
lnLF (ms
2
)
6.9
±
1.1
6.0
±
1.0
0.015
7.6
±
1.1
7.1
±
0.9
0.152
7.6
±
0.9
7.0
±
0.8
0.089
lnHF (ms
2
)
6.5
±
1.5
5.3
±
1.1
0.013
6.8
±
1.4
5.8
±
1.6
0.034
7.2
±
1.3
5.8
±
1.1
0.002
LF/HF
1.4
±
0.8
2.3
±
0.9
0.040
3.0
±
2.3
3.8
±
1.6
0.273
1.9
±
1.5
4.0
±
1.8
0.001
SD1 (ms)
41.9
±
23.2
19.4
±
12.4
0.001 42.9
±
23.4
22.2
±
9.0
0.001
53.6
±
32.7
24.7
±
14.2 0.001
SD2 (ms)
93.7
±
39.9
53.3
±
19.2 < 0.001 120.6
±
39.0
85.0
±
23.9 0.002
144.5
±
49.1
87.7
±
32.6 0.001
SD1/SD2
0.43
±
0.10
0.34
±
0.10
0.018 0.33
±
0.10
0.26
±
0.06 0.005
0.35
±
0.11
0.26
±
0.10 0.071
SampEn
1.6
±
0.2
1.4
±
0.3
0.049
1.4
±
0.2
1.3
±
0.2
0.233
1.3
±
0.2
1.2
±
0.2
0.021
HF: high frequency, HR: heart rate, LF: low frequency, SD: standard deviation, SDNN: standard deviation of the intervals between normal beats, SpO
2
: oxygen satura-
tion, RMSSD: mean square root of successive R-R interval differences, SampEn: sample entropy, TP: total power.