Mountain spa rehabilitation improved health of patients with post-COVID-19 syndrome: pilot study

Abstract

European Association of Spa Rehabilitation (ESPA) recommends spa rehabilitation for patients with post-COVID-19 syndrome. We tested the hypothesis that a high-altitude environment with clean air and targeted spa rehabilitation (MR — mountain spa rehabilitation) can contribute to the improving platelet mitochondrial bioenergetics, to accelerating patient health and to the reducing socioeconomic problems. Fifteen healthy volunteers and fourteen patients with post-COVID-19 syndrome were included in the study. All parameters were determined before MR (MR1) and 16–18 days after MR (MR2). Platelet mitochondrial respiration and OXPHOS were evaluated using high resolution respirometry method, coenzyme Q 10 level was determined by HPLC, and concentration of thiobarbituric acid reactive substances (TBARS) as a parameter of lipid peroxidation was determined spectrophotometrically. This pilot study showed significant improvement of clinical symptoms, lungs function, and regeneration of reduced CI-linked platelet mitochondrial respiration after MR in patients with post-COVID-19 syndrome. High-altitude environment with spa rehabilitation can be recommended for the acceleration of recovery of patients with post-COVID-19 syndrome.

Full text

https://doi.org/10.1007/s11356-022-22949-2
RESEARCH ARTICLE
Mountain spa rehabilitation improved health ofpatients
withpost‑COVID‑19 syndrome: pilot study
AnnaGvozdjáková1 · ZuzanaSumbalová1· JarmilaKucharská1· ZuzanaRausová1· EleonóraKovalčíková2·
TimeaTakácsová2· PlácidoNavas3· GuillermoLópez‑Lluch3· ViliamMojto4· PatrikPalacka5
Received: 4 May 2022 / Accepted: 5 September 2022
© The Author(s) 2022
Abstract
European Association of Spa Rehabilitation (ESPA) recommends spa rehabilitation for patients with post-COVID-19 syn-
drome. We tested the hypothesis that a high-altitude environment with clean air and targeted spa rehabilitation (MR —
mountain spa rehabilitation) can contribute to the improving platelet mitochondrial bioenergetics, to accelerating patient
health and to the reducing socioeconomic problems. Fifteen healthy volunteers and fourteen patients with post-COVID-19
syndrome were included in the study. All parameters were determined before MR (MR1) and 16–18 days after MR (MR2).
Platelet mitochondrial respiration and OXPHOS were evaluated using high resolution respirometry method, coenzyme
Q10 level was determined by HPLC, and concentration of thiobarbituric acid reactive substances (TBARS) as a parameter
of lipid peroxidation was determined spectrophotometrically. This pilot study showed significant improvement of clinical
symptoms, lungs function, and regeneration of reduced CI-linked platelet mitochondrial respiration after MR in patients with
post-COVID-19 syndrome. High-altitude environment with spa rehabilitation can be recommended for the acceleration of
recovery of patients with post-COVID-19 syndrome.
Keywords High-altitude environment, Mountain spa rehabilitation· Post-COVID-19 syndrome· SARS-CoV-2·
Pulmonary function· Clinical symptoms· Platelet mitochondrial metabolism· Coenzyme Q10· Oxidative stress
Responsible Editor: Lotfi Aleya
* Anna Gvozdjáková
anna.gvozdjakova@fmed.uniba.sk
Zuzana Sumbalová
zuzana.sumbalova@fmed.uniba.sk
Jarmila Kucharská
jarmila.kucharska@fmed.uniba.sk
Zuzana Rausová
zuzana.rausova@fmed.uniba.sk
Eleonóra Kovalčíková
riaditel@guhr.sk
Timea Takácsová
takacsova.timka@gmail.com
Plácido Navas
pnavas@upo.es
Guillermo López-Lluch
glopllu@upo.es
Viliam Mojto
viliam.mojto@gmail.com
Patrik Palacka
palacka2@uniba.sk
1 Faculty ofMedicine, Pharmacobiochemical Laboratory
of3rd Department ofInternal Medicine, Comenius
University inBratislava, Sasinkova 4, 81108Bratislava,
Slovakia
2 Sanatorium ofDr. Guhr, 059 81 High Tatras, Tatranská,
Polianka, Slovakia
3 Centro Andaluz de Biología del Desarrollo, Universidad
Pablo de Olavide-CSIC-JA, andCIBERER, Instituto de
Salud Carlos III, Sevilla, Spain
4 Faculty ofMedicine andUNB, 3rd Department ofInternal
Medicine, Derer’s Hospital inBratislava, Comenius
University inBratislava, Limbová 5, 83305Bratislava,
Slovakia
5 Faculty ofMedicine, 2nd Department ofOncology, Comenius
University inBratislava, Klenová 1, 83310Bratislava,
Slovakia
/ Published online: 23 September 2022
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Introduction
The first new coronavirus originated from southeast
China in 2003 (SARS—severe acute respiratory syn-
drome), and the second originated from Middle East
in 2012 (MERS—Middle East respiratory syndrome)
(Hilgefeld and Peiris 2013). In March 11, 2020, the
World Health Organization (WHO) declared a global
pandemic caused by the SARS-CoV-2 beta-coronavi-
rus responsible for a new type of acute respiratory
infection and an atypical pneumonia. WHO named the
diseases caused by SARS-CoV-2 virus as “COVID-
19” (Corona Virus Diseases 2019) (Wu etal. 2020).
Persisting signs or symptoms related to SARS-CoV-2
infection can be divided into two categories. The
first, subacute COVID-19 including symptoms pre-
sent from 4 to 12 weeks beyond acute COVID-19 and
second, post-COVID-19 syndrome (or chronic) includ-
ing symptoms over 12 weeks after the SARS-CoV-2
infection (Fugazzaro etal. 2022). The main symptoms
include shortness of breath, general fatigue, exhaus-
tion, headaches, muscle and joint pain, cough, hair,
taste and smell loss, sleep and memory disturbances,
depression, sensitivity to sound and light, impaired
quality of life and reduced daily activity (35%),
reduced mobility (33%), and pain (33%) (Walle-Hansen
etal. 2021). Taboada etal. (2021) reported limitations
of everyday life near 50% of patients 6 months after
hospitalization for COVID-19. In patients with severe
SARS-CoV-2 infection, dyspnea develops that manifest
as acute coronary distress syndrome (ACDS) and can
lead to death (Wu etal. 2020).
SARS-CoV-2 viral infection occurs with higher inci-
dence in patients with comorbidities such as diabetes
mellitus type 2, obesity, cardiovascular disease, chronic
lung disease, and cancer (Zhang and Liu 2020; Shi etal.
2018; Huang etal. 2020; Li etal. 2020). In aged people,
dysfunctions of immune system and mitochondrial health
are key factors in COVID-19 disease (Lopez-Lluch 2017;
Fernandez-Ayala etal. 2020; Ganji and Reddy 2021).
Mechanical ventilation is required primarily in patients
with comorbidities (Siddiq etal. 2020). In patients with
post-COVID-19 syndrome, individualized rehabilitation
programs are recommended, focused to pulmonary reha-
bilitation of individuals with post-COVID-19 syndrome
(NICE 2022). ESPA, Wang etal. (2020), and Maccarone
and Mesiero (2021) recommend spa pulmonary rehabilita-
tion for patients with post-COVID-19 syndrome.
Virus proteins need mitochondria for their survival
and replication. Mitochondria play the central role in the
primary host defense mechanisms against viral infec-
tions (Gvozdjáková etal. 2020). Many viruses modulate
mitochondrial function, producing more reactive oxygen
species, (ROS), cytokine storm, and stimulate inflamma-
tion (Ganji and Reddy 2021; Gordon etal. 2020). SARS-
CoV-2 infection caused oxidative stress, mitochondrial
dysfunction, platelet dysfunction and coagulation (Ohta
and Nishiyama 2011; Archer etal. 2020), and high mor-
bidity and mortality. SARS-CoV-2 virus may manipulate
mitochondrial dynamics, metabolism, mitochondrial
bioenergetics, apoptosis and antiviral immunity and alter
intracellular distribution of mitochondria.
In 2020, we published the hypothesis that mitochondrial
bioenergetics and endogenous coenzyme Q10 (CoQ10) level
could be targets of the new SARS-CoV-2 virus (Gvozd-
jáková etal. 2020). Currently, this hypothesis was proved
by authors who showed reduced mitochondrial bioenerget-
ics in monocytes (Gibellini etal. 2020) and in peripheral
blood mononuclear cells of patients with COVID-19 (Ajaz
etal. 2021). Our pilot study show reduced platelet mito-
chondrial function with deficit of endogenous CoQ10 level
in non-hospitalized, non-vaccinated patients 3–6 weeks
after acute COVID-19 (Sumbalová etal. 2022). The effect
of SARS-CoV-2 virus on mitochondrial respiratory chain
was named “Mitochondrial COVID-19” (Gvozdjáková
etal. 2022) (Fig.1).
New strategies for COVID-19 prevention and therapy
are being sought to reduce the negative effects of SARS-
CoV-2 virus in society. Environmental strategies play a
vital role in pandemic prevention similar to COVID-19.
Reduction of air quality can support the transmission
dynamics of infectious disease in society with conse-
quential socioeconomic problems (Coccia 2021; Coccia
2021b; Coccia 2022). To the best of our knowledge, the
effect of SARS-CoV-2 and high-altitude environment with
targeted spa rehabilitation on pulmonary function, platelet
mitochondrial bioenergetics, coenzyme Q10 level (CoQ10),
(a key mitochondrial component for energy production),
and lipid peroxidation of patients with post-COVID-19
syndrome has not been described. MR is beneficial for
chronic pulmonary diseases, improving fatigue, joint pain,
psychological stress, sleep disorders, and quality of life in
patients with various diseases (Gvozdjáková etal. 2021).
We tested other hypothesis and strategy for patients
with post-COVID-19 syndrome that a high-altitude envi-
ronment with clean air and targeted spa rehabilitation
of patients with post-C-19 syndrome can contribute to
improving platelet mitochondrial bioenergetics, to accel-
erating patients’ health and to the reducing socioeconomic
problems.
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Materials andmethods
Subjects
The control group (C)
The control group (C) consisted of fifteen healthy individu-
als (6 men and 9 women), aged 38 to 67 years with a mean
age of 51.3 ± 2.3 years, BMI 25.2 ± 0.9 kg/m2. The inclu-
sion criteria for healthy subjects were absence of chronic
medication and no history of COVID-19. Exclusion crite-
ria were lung and heart diseases, diabetes, cancer, obesity,
smoking, and regular alcohol consumption.
The group ofpatients withpost‑COVID‑19 syndrome (MR)
In May and June of 2021, fourteen patients with post-
COVID-19 syndrome, from Sanatorium of Dr. Guhr, High
Tatras, Tatranská Polianka in Slovakia, were included in
this study (MR group—mountain spa rehabilitation). Ten
of them returned questionnaire of clinical symptoms before
and after MR. The group of patients at the time of admission
to mountain spa rehabilitation are marked as MR1 group,
the same group of patients after mountain spa rehabilitation
is marked as MR2 group. The mean age of the patients was
58.69 ± 2.64 years, (8 men and 6 women), BMI 29.85 ±
1.54 kg/m2.
COVID‑19 history ofthepatients withpost‑COVID‑19
syndrome
The patients were hospitalized for three weeks in the period
from November 2020 to April 2021 for COVID-19. The
causes for hospitalization of these COVID-19 patients were
increased body temperature between 37.5 and 39.4°C (n =
8), bilateral pneumonia (n = 9), asthma bronchiale (n = 2),
dyslipoproteinemia (n = 8), and the necessity of oxygen
therapy (n = 8). In the patients, many clinical and psycho-
logical symptoms persisted during next 3–6 months after
hospitalization classified as post-COVID-19 syndrome. The
main symptoms on admission to MR were fatigue, cough,
loss of smell, impaired breathing during exercise, loss of
hair, and depression. In some patients, the loss of appetite
was accompanied with considerable weight loss.
Fig. 1 Effect of SARS-CoV-2
on platelet mitochondrial
respiratory chain and oxidative
phosphorylation in patients
after acute COVID-19. Legend:
SARS-CoV-2 in platelet mito-
chondria of patients after over-
coming the disease COVID-19
decreased the function of
mitochondrial respiratory chain
at complex I, endogenous level
of coenzyme Q10 in Q-CYCLE,
ATP production by oxidative
phosphorylation — Complex
V.; respiratory chain com-
plexes: I, II, III, IV, V; Q-cycle
of coenzyme Q10; cyt c —
cytochrome c; e− — electron;
NADH — reduced nicotinamide
adenine dinucleotide; NAD+ —
nicotinamide adenine nucleo-
tide; FADH2 — flavin adenine
dinucleotide reduced; FAD+ —
flavin adenine nucleotide; O2−
— superoxide radical; H2O2
— hydrogen peroxide; proteins;
lipids, DNA — deoxyribonu-
cleic acid; O2 — oxygen; H2O
— water; ADP — adenosine
diphosphate; ATP — adenosine
triphosphate; Pi — inorganic
phosphate
OUTER
MITOCHONDRIAL
MEMBRANE
INNER
MITOCHONDRIAL
MEMBRANE
II Q
cycle
Cyt C
V
INTERMEMBRANE
SPACE
MATRIX
CELL CYTOPLASM
e-
e-e-
H+
H+
NADH NAD+FADH2FAD+1/2 O2H2O
O2PROTEINS
LIPIDS
DNA
OHH2O2
ADP
+PiATP
IIIIV
e-e-
I
e-
MITOCHONDRIAL COVID-19
(OXPHOS)
PLATELETS
PLATELETS
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Functional capacity ofthelungs
The functional capacity of the lungs was evaluated in ten of
the fourteen patients by 6-min walking test (6MWT) (Brooks
etal. 2002; Casanova etal. 2011), exercise dyspnea during
6MWT by Borg scale (BS) (Borg 1982), and blood oxygen
saturation (SpO2) before and after 6MWT. The results are
summarized in Table1. These tests were performed before
(MR1) and after mountain spa rehabilitation (MR2). Blood
samples were collected the first morning after admission
to the mountain spa before (MR1) and after 16–18 days of
mountain spa rehabilitation (MR2).
Clinical symptoms ofpatients withpost‑COVID‑19
syndrome
Patients completed a questionnaire (21 questions) before and
after MR. The results are summarized in Table2.
Blood count andbiochemical parameters
In all patients with post-COVID-19 syndrome blood counts,
blood lipid parameters, glucose, and CRP were determined
in Hospital of Dr. Vojtech Alexander in Kežmarok, High
Tatras, Slovakia. The determined parameters are summa-
rized in Table3.
Coenzyme Q10 determination
Total coenzyme Q10 concentration (ubiquinol + ubiqui-
none) in whole blood, plasma, and isolated platelets were
estimated using an isocratic HPLC method (Lang etal.
1986; Kucharská etal. 1998). For the oxidation of ubiqui-
nol to ubiquinone, 1,4-benzoquinone was added to blood
or plasma sample (Mosca etal. 2002). The concentrations
of CoQ10-TOTAL were calculated in μmol/L. The isolated
platelets were disintegrated with methanol (Niklowitz etal.
Table 1 Effect of MR on lungs function of patients with post-
COVID-19 syndrome
6MWT 6-min walking text; BS Borg scale; SpO2 blood oxygen satura-
tion; MR1 the patients with post-COVID-19 syndrome at the begin-
ning of the study; MR2 the patients with post-COVID-19 syndrome
after 16–18 days of MR; xp<0.05, xxp<0.01 vs MR1
Parameter MR1 (n = 10) MR2 (n = 10) MR2 vs
MR1 p
value
6MWT (m) 479 ± 40.9 566.2 ± 23.3 0.018x
BS (number) 5.9 ± 0.8 3.8 ± 0.5 0.004xx
SpO2 (%)
Before 6MWT 94.1 ± 0.59 94.1 ± 0.72 ns
After 6MWT 94.9 ± 0.60 93.9 ± 0.78 ns
Table 2 Effect of MR on
clinical symptoms of patients
with post-COVID-19 syndrome
Clinical symptom Before MR (MR1)
(number of symptoms)
After MR (MR2)
(number of symptoms)
Dry cough 3 3
Difficulty breathing 6 3
Shortness of breath in rest 4 3
Elevated temperature 2 0
Chills 2 1
Heart palpitations 3 1
Respiratory support with oxygen 0 0
Weakness 0 0
Overall fatigue 7 2
Malaise 2 2
GIT problems 0 0
Diarrhea 1 1
Chest pain 3 1
Muscle and joint pain 10 5
Back pain 0 0
Headache 4 0
Loss of taste and smell 0 0
Weight loss 1 1
Hearing impairment 2 0
Visual disturbance 3 1
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2004). Concentrations of CoQ10-TOTAL in platelets were cal-
culated in pmol/109 cells.
TBARS
A parameter of oxidative stress — an indicator of lipid per-
oxidation in plasma — was determined spectrophotometri-
cally by measuring the formation of thiobarbituric acid reac-
tive substances (TBARS) (Janero and Bughardt 1989). The
concentration in μmol/L was calculated.
Platelets preparation
Platelets were isolated from whole blood as described pre-
viously (Sumbalova etal. 2018; Palacka etal. 2022) and
counted on hematological analyzer Mindray BC-3600 (Min-
dray, China).
High‑resolution respirometry
The mitochondrial bioenergetics in platelets was evaluated
by high-resolution respirometry (HRR) method (Pesta and
Gnaiger 2012; Sjovall etal. 2013). For the respirometric
assay, 250×106 platelets were used in a 2-mL chamber of
an O2k-Respirometer (Oroboros Instruments, Austria). The
respiration was measured at 37°C in mitochondrial respi-
ration medium MiR05 with addition of 20 mM creatine,
and under continuous stirring at 750 rpm. The data were
collected with DataLab software (Oroboros Instrument,
Austria) using a data recording interval of 2s (Pesta and
Gnaiger 2012; Doerrier etal. 2016). For evaluation of plate-
let mitochondrial bioenergetics, substrate-uncoupler-inhib-
itor (SUIT) protocol 1 (Doerrier etal. 2016) was applied
(Gvozdjáková etal. 2019). The representative trace is in
Fig.2.
The cell suspension volume containing 250 × 106 plate-
lets was added to the 2-mL chamber of O2k-Respirometer
filled with the respiration medium. After stabilization at
ROUTINE respiration of intact platelets, digitonin (0.20
μg/106 cells) was added for plasma membrane permea-
bilization. Next, the chemicals were added in following
order: 1PM–CI-linked substrates pyruvate (5 mM) and
malate (2mM) were added to fuel CI-linked LEAK res-
piration; 2D — saturating ADP (1 mM) was added for
determination of CI-linked respiratory capacity of oxida-
tive phosphorylation (OXPHOS); 2D; c — cytochrome
c (10 μM) was added for testing the outer mitochondrial
membrane integrity; 3U — uncoupler FCCP (0.5 μM)
was added at optimum concentration for determination of
electron transfer (ET) capacity with CI-linked substrates
Table 3 Effect of MR on
blood count and metabolites of
patients with post-COVID-19
syndrome
MR1 The patients before mountain spa rehabilitation; MR2 the patients after mountain spa rehabilita-
tion; WBC white blood cells, RBC red blood cells, HCT hematocrit, PLT platelets, MVC mean corpus-
cular volume, MCH mean corpuscular hemoglobin, MCHC mean corpuscular hemoglobin concentration,
HgB hemoglobin, CHOL total cholesterol, HDL-CH HDL cholesterol, LDL-CH LDL cholesterol, TAG
triacylglycerols, CRP c-reactive protein, GLU glucose. Data are presented as mean ± sem. The differ-
ences between MR1 and the control group, and between MR2 and MR1 group are statistically evaluated,
*p<0.05 vs control, XXp<0.01 vs MR1
Control (n = 15) MRl (n = 14) MR2 (n = 14) MR1 vs C p
value
MR2 vs MR1
p value
Blood count
WBC (109/L) 6.23 ± 0.47 6.99 ± 0.72 6.59 ± 0.64 0.396 0.327
RBC (109/L) 4.66 ± 0.12 4.62 ± 0.12 4.80 ± 0.09 0.717 0.008 xx
HCT (ratio) 0.410 ± 0.100 0.418 ± 0.01 0.438 ± 0.008 0.813 0.003 xx
PLT (109/L) 247.5 ± 16.1 213.9 ± 14.9 219.1 ± 11.2 0.154 0.556
MCV (fL) 87.14 ± 0.65 90.31 ± 1.26 91.21 ± 1.26 0.024* 0.009 xx
MCH (pg) 29.95 ± 0.28 31.58 ± 0.49 31.10 ± 0.41 0.014* 0.079
MCHC (g/L) 343.71 ± 2.53 349.61 ± 2.00 341.08 ± 1.21 0.273 0.002 xx
HgB (g/L) 140.67 ± 3.32 145.46 ± 3.44 149.23 ± 2.89 0.520 0.056
Lipid parameters
CHOL (mmol/L) 5.32 ± 0.27 5.507 ± 0.299 5.76 ± 0.397 0.707 0.264
HDL-CH (mmol/L) 1.41 ± 0.13 1.100 ± 0.086 1.121 ± 0.099 0.031* 0.632
LDL-CH (mmol/L) 3.09 ± 0.25 3.368 ± 0.287 3.344 ± 0.316 0.319 0.904
TAG (mmol/L) 2.05 ± 0.49 2.489 ± 0.555 3.224 ± 0.954 0.055 0.142
Other parameters
CRP (mg/L) 0.90 ± 0.20 1.80 ± 0.45 1.81 ± 0.53 0.721 0.950
GLU (mmol/L) 5.13 ± 0.17 6.17 ± 0.63 5.20 ± 0.26 0.139 0.069
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pyruvate+malate; 4G — glutamate (10 mM) was added
for evaluation of ET capacity with CI-linked substrates
pyruvate+malate+glutamate; 5S — CII-linked sub-
strate succinate (10 mM) was added for determination
of CI&CII-linked ET capacity. For evaluation of mito-
chondrial pathway–related rates (here labeled according
to titration steps), the rate after digitonin representing
residual oxygen consumption (ROX) was subtracted from
all respiratory rates (Fig.2).
Citrate synthase
The activity of citrate synthase as mitochondrial marker was
evaluated spectrophotometrically according to the method of
Srere (1969a, 1969b), described in detail by Eigentler etal.
(2020). The activity of CS is evaluated in μmol/min/106
cells.
Data analysis
The differences between parameters of the post-COVID-19
MR1 group and the control group were evaluated using
unpaired Student’s t test. For evaluation, the difference
between MR1 and MR2 paired Student’s t test was used. P
values <0.05 were considered statistically significant. The
results are shown as individual points and the mean ± stand-
ard error of mean (sem).
Results
Pulmonary function of patients with post-COVID-19 syn-
drome was evaluated by 6-min walking test (6MWT), exer-
cise dyspnea by Borg scale (BS), and blood oxygen satu-
ration (SpO2). By 6MWT, the distance that a patient can
quickly walk in a period of 6 min is measured, reflecting
the functional pulmonary capacity. In our patients, 6MWT
test improved significantly after MR (from 479 ± 40.9 m
to 566.2 ± 23.3 m, p = 0.018), the walked distance dur-
ing the 6MWT increased by 87.2 m. Exercise dyspnea was
measured by BS points from 0 to 10. Zero on BS means
no dyspnea and 10 points on BS reflect maximal dyspnea
after 6MWT. Exercise dyspnea measured by BS statistically
significantly improved in patients with post-COVID-19 syn-
drome after MR by 2.1 points (from 5.9 ± 0.8 points to 3.8
± 0.5 points, p = 0.004). Physiological levels of SpO2 are
between 95 and 100%. SpO2 before 6MWT and after 6MWT
were without significant changes after MR (Table1).
Effect ofMR onclinical symptoms ofpatients
withpost‑COVID‑19 syndrome
From fourteen patients, ten patients filled out the question-
naire for evaluation of clinical symptoms before and after
MR. Several patients had more than three clinical symp-
toms of COVID-19 before MR. Many clinical symptoms
have improved after MR, as breathing difficulty, shortness
of breath, chills, heart palpitations, overall fatigue, muscle
Fig. 2 The trace from the measurement of platelet mitochondrial
respiration in freshly isolated platelets (Doerrier et al. 2016). Leg-
end: The blue line shows oxygen concentration (μM) and the red
trace oxygen consumption (pmol O2/s/106 cells). 250 × 106 platelets
were added into a 2-mL chamber of an O2k-Respirometer with mito-
chondrial respiration medium MiR05 plus 20 mM creatine at 37 °C
and continuous stirring (750 rpm). The titration steps are cells (ce),
digitonin (Dig); pyruvate plus malate (PM); adenosine diphosphate
(ADP); cytochrome c (cyt c); uncoupler FCCP (U); glutamate (G);
and succinate (S). All substrates were added in kinetically saturating
concentrations; FCCP was titrated in optimum concentration to reach
the maximum O2 flow. ce — intact cells; ROX — residual oxygen
consumption; CI — complex I pathway; CI&II — complex I and
complex II pathway; LEAK — non-phosphorylating resting state of
respiration (L); OXPHOS — the phosphorylating state of respiration
(P); ET — noncoupled state of respiration at optimum concentration
of uncoupler
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and joint pain, chest pain, headache, hearing impairment,
and visual disturbance (Table2).
Effect ofMR onblood count andmetabolites
ofpatients withpost‑COVID‑19 syndrome
MR significantly improved blood count, as the count of RBC
(p = 0.008), HCT (p = 0.003), MCV (p = 0.009), and HgB
(p = 0.056) were higher in MR2, and MCHC was lower
(p = 0.002) compared to MR1. Mean of lipids parameters
(CHOL, HDL-CH, LDL-CH, TAG) of post-COVID-19
patients showed dyslipoproteinemia. These parameters were
not influenced by mountain spa rehabilitation (Table3). CRP
was higher in patients with post-COVID-19 syndrome vs
control and did not improve after MR. Slightly higher blood
glucose level of the patients improved after MR (p = 0.069,
Table3).
Effect ofMR onimpaired platelet mitochondrial
bioenergetics inpatients withpost‑COVID‑19
syndrome
We used freshly isolated platelets from patients with post-
COVID-19 syndrome before MR (MR1) and after 16–18
days of special MR (MR2). All platelet respiratory param-
eters are expressed as JO2/CS (pmol/s/IU). The results of
platelet mitochondrial bioenergetics analysis are shown in
Fig.3 and in supplementary material Fig. S3A-S3H.
Detailed results are shown in supplementary material,
Fig. S3A–S3H. Routine respiration of intact platelets (ce)
was similar in all groups (Fig.3, Fig. S3A). The rate of
mitochondrial LEAK respiration with CI-linked substrates
(1PM) in MR1 group was lower (by 14.2%), although not
statistically significant in comparison with control group.
In MR2 group, mitochondrial LEAK respiration with CI-
linked substrates was significantly increased vs MR1 (by
47.8%, p = 0.029, Fig.3; Fig. S3B). CI-linked respiration
coupled with ATP production (2D — CI-linked oxidative
phosphorylation (OXPHOS) capacity) in the MR1 group
was significantly reduced (p = 0.0004) by 45.8% vs con-
trol group values. In MR2 group, this parameter associated
with ATP production was slightly improved (by 12.3% vs
MR1) (Fig.3; Fig. S3C). The respiration after addition of
cytochrome c (2D;c) in the MR1 group was decreased by
50.6% vs control group values (p = 0.00002), in MR2 group,
this parameter was slightly improved vs MR1 group (by
15.3%) (Fig.3; Fig. S3D). Maximal mitochondrial oxidative
capacity (the electron transfer capacity, ET) after uncoupler
titration (3U) was significantly reduced in MR1 group vs
control group (by 45.7%, p = 0.0002). In MR2 group, this
parameter was slightly improved vs MR1 (by 8.8%) (Fig.3;
Fig. S3E). After addition of CI-linked substrate glutamate
(4G), the ET capacity was significantly lower in MR1 group
vs control group (by 40.0%, p = 0.0005). This respiration
was slightly increased in MR2 group vs MR1 (by 15.6%,
Fig.3; Fig. S3F). ET capacity with CI&II-linked substrates
(5S) was lower in MR1 group vs control group (by 9.7%, p =
0.060). This parameter was slightly higher in MR2 vs MR1
group (by 4.8%, Fig.3; Fig. S3G). The mean improvement
of mitochondrial parameters representing OXPHOS and ET
capacity was 11.4% (from 4.8 to 15.6%) in comparison with
MR1 group, which was taken as 100% (Fig.3; Fig. S3C
– S3G).
The mitochondrial marker — the activity of citrate syn-
thase — was increased in patients with post-COVID-19
syndrome in comparison with control group (by 34.7%, p =
0.0004). After MR, the activity of citrate synthase in plate-
lets slightly decreased vs MR1 (by 10.8%, p = 0.092, Fig.4).
Effect ofMR onTBARS andendogenous CoQ10
inpatients withpost‑COVID‑19 syndrome
There was no significant difference in TBARS concentration
between control group and patients with post-COVID-19
syndrome. Endogenous concentration of CoQ10-TOTAL
(ubiquinone + ubiquinol) in platelets, blood, and plasma of
the post-COVID-19 syndrome group did not significantly
Fig. 3 Effect of mountain with spa rehabilitation on platelet mito-
chondrial bioenergetics in patients with post-COVID-19 syndrome.
Legend: ce: ROUTINE respiration of intact platelets; 1PM: com-
plex I-linked LEAK (state 4) respiration with substrates (pyru-
vate + malate); 2D: complex I-linked OXPHOS (state 3) respira-
tion capacity associated with CI-linked ATP production; 2D;c: The
OXPHOS capacity after cytochrome c addition; 3U: The respiration
after uncoupler FCCP titration represents CI-linked electron trans-
fer (ET) capacity with substrates pyruvate+malate; 4G: ET capacity
with substrates pyruvate+malate+glutamate; 5S: CI&CII-linked ET
capacity with substrates pyruvate + malate + glutamate + succinate,
(Doerrier etal. 2016; Gvozdjáková etal. 2019). The respiratory rates
are marked according the steps in the SUIT protocol 1 (see Fig.2).
Control — the control group; MR1 — patients before mountain spa
rehabilitation; MR2 — patients after mountain spa rehabilitation. CI
— complex I pathway; CI&CII — complex I and complex II path-
way; LEAK — the non-phosphorylating resting state of respiration;
OXPHOS — the phosphorylating state of respiration; ET — the non-
coupled state of respiration at optimum uncoupler concentration
14206 Environmental Science and Pollution Research (2023) 30:14200–14211
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Content courtesy of Springer Nature, terms of use apply. Rights reserved.

differ from the control group and did not change after MR
(Table4).
Discussion
WHO recommended rehabilitation (WHO 2022) and ESPA
recommended spa rehabilitation of the patients with post-
COVID-19 syndrome. The rehabilitation improved pul-
monary function, exercise capacity, and quality of life of
patients with post-acute phase of COVID-19 (Nalbandian
etal. 2021; Schaeffer etal. 2022). Other authors used reha-
bilitation based on respiratory physiotherapy techniques, on
exercise training or in combination with yoga (Srinivasan
etal. 2021; Herrera etal. 2021).
The current pilot study was undertaken to determine
the effect of high-altitude environment and targeted reha-
bilitation in spa on pulmonary function, clinical symp-
toms, endogenous coenzyme Q10 levels, oxidative stress,
and platelet mitochondrial oxidative phosphorylation
(OXPHOS) function in patients with post-COVID-19
syndrome. In high-altitude environment of High Tatras
in Slovakia, spa rehabilitation in Sanatorium of Dr. Guhr,
Tatranská Polianka is used for curing chronic pulmonary
diseases for many years. The Sanatorium is located at alti-
tude of 1005 m above sea level, in the zone of forests with
dry air, favorable solar radiation, reduced partial oxygen
pressure and air pressure, and with a mild, relatively stable
daily temperature (Gvozdjáková etal. 2019). For patients
with post-COVID-19 syndrome in high-altitude environ-
ment and spa rehabilitation program include walking,
breathing exercises, oxygen therapy, exercise, water pro-
cedures, massages, psychological support, and education
(Jendrichovsky etal. 2021; Tiku etal. 2020). The reha-
bilitation program is individualized for the improvement
of mental health, to prevent skeletal muscle hypotrophy
with a focus on increasing the rate of daily movement and
overall patient activity.
Beneficial effect of pulmonary rehabilitation was docu-
mented in patients with chronic respiratory disease (Spruit
etal. 2013). Improvements in exercise capacity, dyspnea,
fatigue, anxiety, and depression after a pulmonary rehabilita-
tion were reported by Soril etal. (2022). We evaluated pulmo-
nary function of patients with post-COVID-19 syndrome by
6MWT, BS, and SpO2. By 6-min walking test, the distance
that a patient can quickly walk in a period of 6 min (6MWT)
is measured, reflecting the functional pulmonary capacity
(Brooks etal. 2002; Casanova etal. 2011). After MR2 6MWT
improved significantly (p = 0.018), the walking distance
increased by 87.2 m. The increase in 6MWT by 70 m is con-
sidered clinically important for patients. Exercise dyspnea was
evaluated by Borg scale (Borg 1982). After MR2, the exercise
dyspnea was significantly improved (p = 0.004). An improve-
ment by 0.5 point on BS is considered as an improvement of
lung function. Oxygen saturation (SpO2) levels before and after
6MWT were without significant changes after MR (Table1).
Rehabilitation of patients in a high-altitude environment
reduced the extent of physical, cognitive, and mental impair-
ment (as breathing, total fatigue, muscle, joint and chest
pain, headache, memory impairment, depression, hearing
impairment, visual disturbances), and improved the quality
of life of patients with post-COVID-19 syndrome (Table2).
Although special rehabilitation in the Sanatorium lasted only
16–18 days, the positive effect of MR was manifested in
patients with post-COVID-19 syndrome.
Fig. 4 Effect of MR on citrate synthase activity in platelets of patients
with post-COVID-19 syndrome. Legend: CS — citrate synthase;
MR1 — before mountain spa rehabilitation; MR2 — after mountain
spa rehabilitation
Table 4 Effect of MR on lipid
peroxidation and CoQ10-TOTAL
concentration of patients with
post-COVID-19 syndrome
TBARS indicator of lipid peroxidation; CoQ10-TOTAL ubiquinol + ubiquinone; MR1 before mountain spa
rehabilitation; MR2 after mountain spa rehabilitation;
Parameter Control (n = 15) MR1 (n = 14) MR2 (n = 14)
TBARS in plasma (μmol/L) 4.80 ± 0.18 4.65 ± 0.16 4.52 ± 0.17
CoQ10-TOTAL in:
Platelets (pmol/109 cells) 84.14 ± 5.56 93.92 ± 5.92 91.47 ± 7.11
Blood (μmol/L) 0.313 ± 0.020 0.366 ± 0.035 0.315 ± 0.017
Plasma (μmol/L) 0.516 ± 0.032 0.516 ± 0.045 0.509 ± 0.035
14207Environmental Science and Pollution Research (2023) 30:14200–14211
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Several pathobiochemical mechanisms participate in
virus infection on cellular and subcellular level. Mitochon-
dria (subcellular particles) play a central role in the primary
host defense mechanisms against viral infections, and in
these processes, a number of novel viral and mitochondrial
proteins are involved. One possible mechanism of SARS-
CoV-2 effect is a manipulation of mitochondrial bioenerget-
ics indirectly, by ACE2 regulation, and the other possibility
is manipulation by localizing ORF-9b (open reading frame)
protein to mitochondria. Manipulations of host mitochon-
dria by viral ORFs can release mtDNA in the cytoplasm,
activate mtDNA-induced inflammasome, and suppress
innate and adaptive immunity (Singh etal. 2020; Singh
etal. 2021). In the pathological conditions, as by virus acti-
vated cells, their request for energy production is increased
(Singh etal. 2020). Under physiological conditions, plate-
lets receive approximately 60% of energy from glycolysis
and 40% energy from OXPHOS (Gatti etal. 2020; Warburg
etal. 1927). Other mechanism of SARS-CoV-2 virus is its
role in manipulating mitochondrial function. SARS-CoV-2
hijacks of host mitochondria of immune cells in COVID-
19 disease (Singh etal. 2020), and impairs mitochondrial
dynamics leading to cell death (Ganji and Reddy 2021; Seth
etal. 2005). Mitochondrial “hijacking” by SARS-CoV-2
virus could be a key factor in the pathogenesis of this virus
and induction of COVID-19 (Saleh etal. 2020; Singh etal.
2021). A good mitochondrial fitness could be considered
as a protective factor against viral infections, including
COVID-19 (Maccarone and Mesiero 2021; Burtscher etal.
2021; Jimeno-Almazán etal. 2021).
Mitochondrial antiviral signaling protein (MAVS), asso-
ciated with the outer mitochondrial membrane, mediates
the activation of NFK-B and the induction of interferons in
response to viral infection (Sun etal. 2006; Seth etal. 2005).
Many viruses target mitochondrial metabolism, dynamics,
mitochondrial bioenergetics, membrane potential, ion per-
meability, induce reactive oxygen species production, alter
the Ca2+ regulatory activity, and cause oxidative stress in
host cells (Anand and Tikoo 2013; Elesela and Lukacs
2021). Viruses can modulate apoptosis and mitochondrial
antiviral immunity, alter intracellular distribution of mito-
chondria, cause host mitochondrial DNA depletion for their
survival in the cell (Ohta and Nishiyama 2011; Ripoli etal.
2010; Di Gennaro etal. 2020). Progression of the disease
in COVID-19 patients involves “cytokine storm” with iron
dysregulation (as hyperferritinemia) which induces ROS
production and oxidative stress (Saleh etal. 2020).
The regeneration of mitochondria impaired by SARS-
CoV-2 viruses can be achieved by various means including
breathing exercises, increased physical activities, reduction
of daily calories intake, enhanced daily intake of food with
antioxidants properties (Ganji and Reddy 2021), spa rehabili-
tation (Wang etal. 2020; Maccarone and Mesiero 2021), and
targeted coenzyme Q10 supplementation (Gvozdjáková etal.
2019). This pilot study showed significant deficit of platelet
mitochondrial complex I-linked ET capacity and OXPHOS
respiration associated with ATP production in patients with
post-COVID-19 syndrome which were improved by spa
rehabilitation in a high-altitude environment.
An essential component of the mitochondrial respira-
tory chain for energy (adenosine triphosphate) production
is coenzyme Q10 (CoQ10) with antioxidant properties. In
physiological conditions, CoQ10 transports electrons from
complex I and complex II to complex III. Complexes of
respiratory chain (CI, CIII, and IV) are organized in super-
complexes minimizing the distance for electron transfer. In
the pathological conditions, electron flux from CoQ can be
reversed to CI reducing NAD+ — the phenomenon known as
the reverse electron transfer (RET) (Hidalgo-Gutiérrez etal.
2021; Scialo etal. 2017). We suppose that impaired platelet
mitochondrial metabolism in patients with post-COVID-19
syndrome can contribute to the reprogramming of mitochon-
drial OXPHOS toward glycolysis.
Viral infections induce production of reactive oxygen spe-
cies, which can contribute to the alterations of mitochon-
drial bioenergetics. Different viruses are able to modulate
antioxidant enzymes (Singh etal. 2021; Hidalgo-Gutiérrez
etal. 2021). In our patients, the endogenous CoQ10 levels
and TBARS in plasma of patients with post-COVID-19 syn-
drome were similar to control data, probably as a result of
therapy with oxygen and drugs with antioxidant properties
before starting MR.
High-altitude of the mountain spa environment improved
mitochondrial fitness as could be seen from improved CI-
linked OXPHOS and ET capacity of platelet mitochondria
of patients with post-COVID-19 syndrome (Fig.3, Fig.
S3E – S3G). In MR2 group, platelet mitochondrial CI-
linked LEAK respiration (L) was significantly increased
vs MR1 (Fig.3, Fig. S3B). The parameter P-L control effi-
ciency (Gnaiger 2020) calculated from ADP-stimulated and
LEAK reaspiration as (P-L)/P was slightly lower in the MR1
group vs controls, and after MR declined by 9.5% vs MR1
(p = 0.055) (Fig.3, Fig. S3H). This parameter with values
from 0 to 1 is a measure of coupling control efficiency. The
mechanisms leading to decreased P-L control efficiency
after MR in patients with post-COVID-19 syndrome could
be a matter of further research. It could be speculated that
increased physical activity in MR could induce oxidative
stress mediating higher proton conductance of inner mito-
chondrial membrane at high proton motive force (at LEAK
state), preventing this way increased ROS production by
platelet mitochondria. An increase in LEAK respiration and
a decrease in P-L control efficiency was found in platelets
of ultramarathon runners after the race, reflecting increased
proton leakage across the inner mitochondrial membrane
(Hoppel etal. 2021). The increased CS activity in platelets
14208 Environmental Science and Pollution Research (2023) 30:14200–14211
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Content courtesy of Springer Nature, terms of use apply. Rights reserved.

of patients with post-COVID-19 syndrome may indicate
increased density of mitochondria as a compensation for
their decreased function.
Conclusions
Comprehensive strategy for virus pandemic has to be based
on medical evidence, on effective vaccines to decrease
mortality, to improve economic growth and socioeconomic
system. Spa rehabilitation in high-altitude environment
contributes to the acceleration of patients’ health and to the
reduction of socioeconomic problems. Our pilot findings
contribute to the understanding of the role of mitochondria
in the pathogenesis of COVID-19. Mountain spa rehabilita-
tion can be recommended for the acceleration of recovery
of patients with post-COVID-19 syndrome.
Limitations of these pilot results include relatively short
time of mountain spa rehabilitation (16–18 days) paid by
the insurance company and number of patients with post-
COVID-19 syndrome (n = 14).
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s11356- 022- 22949-2.
Acknowledgements We thank the National Institute for Pediatric Res-
piratory Diseases and Tuberculosis, n.o., Dolný Smokovec, Slovakia,
for collaboration in counting of isolated platelets. Technical assistance,
Anna Štetková and Jana Bertalanová, from Pharmacobiochemical Lab-
oratory of 3rd Department of Internal Medicine, Faculty of Medicine,
Comenius University in Bratislava, Slovakia.
Author contribution Anna Gvozdjáková and Eleonóra Kovalčíková
designed the study. The first draft of the manuscript was written by
Anna Gvozdjáková, and all authors commented of previous versions of
the manuscript. Patrik Palacka and Timea Takácsová collected clinical
data. Zuzana Sumbalová and Zuzana Rausová measured and evalu-
ated platelet mitochondrial function; Jarmila Kucharská measured and
evaluated antioxidants. Zuzana Sumbalová performed the statistical
analysis and created figures. Viliam Mojto reviewed the manuscript.
Plácido Navas and Guillermo López-Lluch reviewed and completed
the manuscript. All authors read and approved the final manuscript.
Funding This research was partially funded by the Comenius Univer-
sity in Bratislava, Faculty of Medicine and by OncoReSearch, Rovinka,
Slovakia.
Data availability The supporting data are available from the authors
upon request.
Declarations
Institutional review board statement The study was carried out accord-
ing to the principles expressed in the Declaration of Helsinki (World
Medical Association 2022), and the study protocol was approved by
the Ethics Committee of Dérer’s Hospital in Bratislava, Limbová 5, 833
05 Bratislava, Slovakia, Code: 12/2021. The randomized controlled
clinical trials registration number is NCT05178225.
Consent to participate Written informed consent form was obtained
from each participant before including to the study group.
Consent for publication The authors declare that they have all rights
to publish the presented anonymous data.
Competing of interest The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article's Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
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