Effects of acetazolamide combined with remote ischemic preconditioning on risk of acute mountain sickness: a randomized clinical trial

This is a single-center, prospective, randomized, open-label, blinded endpoint (PROBE) study. Participants were randomly assigned to five groups with different interventions: Acetazolamide group, Regular RIPC Training (Ripc) group, Rapid RIPC Training (Rapid-Ripc) group, Acetazolamide plus Rapid RIPC Training (Combined) group, and Control group. After the interventions, participants were exposed to a normobaric hypoxic chamber for 6 h (12% oxygen/88% nitrogen, approximately equivalent to an altitude of 4000 m after adjusting the difference between the actual altitude and the stimulated altitude in a normobaric hypoxic chamber [19]). The primary and secondary outcomes were assessed at baseline, before and after the 6 h of hypoxic exposure. We conducted the study in five rounds due to the large number of participants. Each round lasted for 1 week with 50 participants, with an overall study lasting from Dec. 2021 to Aug. 2022.

We recruited healthy lowlander volunteers aged 18 to 50 years without a prior history of high-altitude exposure (> 1500 m) within a month. The exclusion criteria included the following: (1) having any acute or chronic physical or mental disease (including hypertension, diabetes mellitus, coronary heart disease, chronic obstructive pulmonary disease, cerebrovascular disease, migraine, anxiety disorder, depression, and insomnia), (2) peripheral oxygen saturation (SpO₂) < 95%; (3) having any contradiction for RIPC (including severe soft tissue injury, fracture, or peripheral vascular disease in the upper limbs), (4) allergy to sulfonamides, (5) previous laboratory tests suggesting hypokalemia, hyponatremia or hepatic or renal impairment, (6) taking one or more medications regularly daily, (7) having a smoking or heavy drinking habit, (8) being pregnant or breastfeeding (women), (9) being enrolled or having been enrolled in another clinical trial within three months of this clinical trial.

Sample size was calculated based on previous data. The incidence of AMS at 4000 m altitude without any intervention was about 40% [2, 3]. A study on RIPC for the prevention of AMS showed that a 1-week course of RIPC reduced the incidence of AMS at 3650 m by 25%, from 40 to 30% [15]. In addition, systematic reviews showed that acetazolamide could decrease the incidence of AMS by 47% [6], which corresponded to an incidence of 21% at 4000 m altitude. Therefore, theoretically, the incidence of AMS after using the combination of the two methods was about 16%. With a 5% significance level and 80% power, the sample size of this study included 50 participants in each group, a total of 250 participants.

Participants were randomized in a 1:1:1:1:1 ratio via simple random sampling. Each participant drew a small ball from a box that contained identically shaped, sized, and colored balls, which were evenly mixed. Each ball was labeled with a number ranging from 001 to 250. Participants with end number zero or five were assigned to Control group, one or six to Combined group, two or seven to Ripc group, three or eight to Rapid-Ripc group, and four or nine to Acetazolamide group.

At baseline, a comprehensive clinical assessment, including consultation and physical examination, was performed for each participant by physicians at Xuanwu Hospital, Capital Medical University in Beijing, China (40 m above sea level). Participants were then randomly assigned to five groups to receive different interventions. The RIPC training was induced by Renqiao Remote Ischemic Conditioning Device. Each training comprised five cycles of bilateral upper limb ischemia and reperfusion, which was induced by two cuffs placed around the upper arms respectively and inflated to a pressure of 180 mmHg for 5 min followed by 5 min of reperfusion by cuff deflation. The protocol was as follows:

Participants assigned to Control group did not receive specific intervention before entering the hypoxic chamber.

The intervention process was conducted at Beijing Highland Conditioning Medical Center with a dedicated supervision team to ensure completion of the intervention. All participants taking acetazolamide were required to visit the site at 8 am and 4 pm to take their medication, under the supervision of the research physician responsible for dispensing and recording. Participants for the twice-daily RIPC training also came to the site for morning and afternoon training sessions. For the four-times-daily RIPC training, three sessions were conducted on-site, with participants bringing the device home for the final session and providing training photos to the research physician. These photos included photos of the training device screen for each round of inflation-deflation, photos of upper extremities engorgement at the end of the session, and photos of the training environment. The research physician verified and recorded participant’s compliance with the trial protocol. All participants completed the intervention for the trial as required.

After the interventions, participants entered the hypoxic chamber at Beijing Highland Conditioning Medical Center and stayed from 9 am to 3 pm. During the hypoxic exposure, participants stayed seated and performed some low physical activities (e.g., standing, walking, and playing card games). The physician assessed the participants’ vital signs hourly (including blood pressure, heart rate, and SpO₂). All the participants were encouraged to persist to the end of the 6 h of hypoxia. If severe intolerable symptoms did occur, or if vital signs became exceedingly abnormal (any of the following: SpO₂ < 70% [20, 21], systolic blood pressure > 160 or < 90 mmHg, diastolic blood pressure > 100 or < 60 mmHg, heart rate > 75% × (220-age) or < 60 beats per minute, respiratory rate > 30 or < 12 breaths per minute), the participant exited the hypoxic room and received oxygen therapy.

The primary outcomes included the incidence and severity of AMS, and SpO₂ after hypoxic exposure. The incidence of AMS was evaluated by the 2018 Lake Louise Acute Mountain Sickness Score at the end of the 6 h of hypoxic exposure. A positive AMS was defined as a total score of the four symptoms (headache, gastrointestinal symptoms, fatigue and/or weakness, and dizziness/light-headedness) of at least three points with a headache score of at least one point. The severity of AMS was evaluated by the 2018 Lake Louise Acute Mountain Sickness Score, Acute Mountain Sickness-Cerebral score and Chinese Acute Mountain Sickness score. The participants completed the questionnaires independently. SpO₂ was measured by finger pulse oximetry (Yuyue Medical Equipment Co., Ltd, Shanghai, Beijing) after hypoxic exposure. Secondary outcomes included systolic and diastolic blood pressure and heart rate after hypoxic exposure. Exploratory outcomes included the hematological parameters evaluated at baseline, pre-hypoxia and post-hypoxia, including venous blood gas, complete blood count (post-hypoxia), human cytokine antibody array evaluation, ELISA validation, and genetic polymorphisms analysis, to explore the mechanisms of the combined regimen. Adverse reactions of acetazolamide or RIPC were also assessed. All data and analyses for the primary and secondary outcomes were assessed by investigators blinded to treatment allocation. A blinded-to-treatment adjudicating group reviewed the data to determine which participants had reached study outcomes and to evaluate safety.

Venous blood samples were drawn at baseline, pre-, and post-hypoxia. During each blood collection, 2 ml venous samples were collected by heparin-coated syringe and analyzed immediately using Vitagas 8E (Kangli Biomedical Inc., Shenzhen, China); 5 ml was collected by Ethylenediamine Tetraacetic Acid (EDTA)-coated vacutainers, and centrifuged at 3000 rpm for 15 min. The plasma was stored at -  80 °C. At post-hypoxia, an additional vacutainer of blood was drawn from all of the participants for a complete blood count test.

During the first round of the study, twenty-four participants were randomly selected from the Control, Acetazolamide, Rapid-Ripc, and Combined group (n = 6 for each group) and their samples at baseline, pre-, and post-hypoxia were analyzed for 440 cytokines using the human cytokine antibody array (RayBiotech Inc., Norcross, GA, USA) as described in the manufacturer’s protocol. The 440 cytokines primarily encompass inflammation, angiogenesis, cell migration, cell proliferation, autophagy, apoptosis, oxidative stress, atherosclerosis, cancer-related pathways, etc. The raw fluorescence signal data obtained from the experiments were normalized, compared between groups using moderated t-statistics (data were processed in R/Bioconductor). Differentially expressed proteins (DEPs) were identified based on a fold change > 2 or < 0.5, and P value < 0.05. Protein-protein association network was investigated by STRING analysis (http://​string-db.​org). Based on the literature review, protein signal intensity, and kit accessibility, PDGF-AB was selected and quantitatively validated using ELISA kits (RayBiotech Inc., Norcross, GA, USA) in all the participants, following the manufacturer’s protocol.

Gene polymorphisms of PDGFA and PDGFB were detected in all subjects. We searched for potentially regulatory single-nucleotide polymorphisms (SNPs) based on previous researches and National Center for Biotechnology Information (NCBI) SNP database (http://​www.​ncbi.​nlm.​nih.​gov/​projects/​SNP/​). Selected SNPs were required to have a minor allele frequency (MAF) > 0.1 and have been previously studied or reported to be correlated with PDGF protein concentrations. For PDGFA, we selected rs1800814 [22], rs2070958 [22], rs9690350 [23], and rs62433334 [22, 24] in introns. For PDGFB, we selected rs1800817 [25‐27], rs2285099 [28], and rs2285094 [29] in introns, and rs1800818 [25‐27, 30] at 5′-UTR.

Genomic DNA was extracted from whole blood following the instructions provided with the Finemag Blood DNA kit (Genfine Biotech Co. Ltd., Beijing, China). The extracted genomic DNA samples were stored at - 20 °C until analysis. The polymerase chain reaction (PCR) primers are listed in Additional file 1: Table S1. Following PCR amplification and purification, the DNA sequences were analyzed using the ABI 3730xl DNA Analyzer. The assays were repeated for 5% of the samples, and the results were 100% concordant. Investigators involved in SNP analyses were blinded to participants’ clinical data.

Continuous variables were expressed as means with standard deviation (SD). Normality of distribution for continuous variables was assessed using the Shapiro-Wilk test. Categorical variables were expressed as counts and percentages. For the primary outcomes, the planned comparison of the incidence of AMS between groups was conducted using χ² test. The risk ratio with confidence intervals was reported. The AMS score between groups were investigated using one-way ANOVA or Kruskal-Wallis test. The comparisons of SpO₂ after hypoxic exposure were conducted using paired two-way ANOVA (different time point after hypoxia vs different intervention groups). For secondary outcomes, the systolic and diastolic blood pressure and heart rate after hypoxic exposure were investigated using paired two-way ANOVA. For exploratory outcomes, we pre-specified the comparisons of venous blood gas parameters after interventions using paired two-way ANOVA. We pre-specified the cytokine antibody research in each of Control, Rapid-Ripc, Acetazolamide, and Combined group before and after hypoxia. After identifying PDGF-AB, PDGF-AB levels were validated and investigated using paired two-way ANOVA, and means with SD and mean differences with their 2-sided 95% confidence interval (CI) were reported. We further divided the participants into subgroups based on AMS (+)/(-) and based on different intervention groups to analyze the change of PDGF-AB. Analyses were conducted with GraphPad Prism version 9.0 (GraphPad Software: San Diego, CA, USA). Turkey’s multiple comparisons were performed between each pair of groups.

Genetic data of PDGF were tested for Hardy–Weinberg equilibrium using χ²-test. Genotypes were analyzed in codominant, dominant, recessive, and log-additive model using SNPStats online software (http://​bioinfo.​iconcologia.​net/​SNPstats). Allele frequencies and genotype distribution were compared between AMS (+) and AMS (-) groups using the χ²-test. Binary logistic regression was used to analyze associations between genotypes and AMS, which were further adjusted for age, sex, and body mass index (BMI). We divided the participants into subgroups based on their genotype and interventions, and analyzed the PDGF-AB levels in different subgroups. The level of significance was set as a 2-sided P value less than 0.05.

The study was approved by the Ethics Committee of the Xuanwu Hospital, Capital Medical University and registered on ClinicalTrials.gov (NCT05023941). All participants provided written informed consent. The study was conducted in compliance with the Declaration of Helsinki and Good Clinical Practice guidelines.

From 279 subjects who signed up and met the enrollment criteria, 252 were randomized to different groups. Two terminated the study prematurely during the intervention phase. Thus, a total of 250 participants finally entered the hypoxic chamber and were included in the analysis, among them two left the chamber at 2-h or 5-h hypoxic exposure due to severe discomfort (Fig. 2). The participants consisted of 152 (60.8%) women and 98 (39.2%) men, with a mean age of 30.38 ± 7.17 years. The baseline characteristics are presented in Additional file 1: Table S2.

A total of 48 (19.2%) participants developed AMS in our study. Combined group exhibited the lowest incidence and mildest severity of AMS. The number of AMS-positive participants in Control, Ripc, Rapid-Ripc, Acetazolamide, and Combined group were 13 (26%), 11 (22%), 14 (28%), 7 (14%), and 3 (6%), respectively (P = 0.030, Fig. 3a). Statistical differences were found between Combined and Control (RR 0.23, 95% CI 0.07 to 0.70, P = 0.006), Ripc (RR 0.27, 95% CI 0.09 to 0.84, P = 0.021), and Rapid-Ripc group (RR 0.21, 95% CI 0.07 to 0.64, P = 0.003). No significant difference was observed between Acetazolamide and the other groups (vs Control, P = 0.134; vs Ripc, P = 0.298; vs Rapid-Ripc, P = 0.086; vs Combined, P = 0.183).

The Lake Louise AMS Score of the five groups after hypoxic exposure were 1.68 ± 1.71, 1.72 ± 1.96, 1.72 ± 1.40, 1.36 ± 2.11, and 0.92 ± 1.10, respectively (Fig. 3b). Combined group exhibited lower scores than the other groups, and the difference was statistically significant when compared to Rapid-Ripc group (P = 0.034). The AMS-Cerebral Score and the Chinese AMS score also showed that Combined group was less symptomatic, but there were no significant differences between the groups (Additional file 1: Figure S1). We further analyzed the distribution of AMS scores in five groups (Fig. 3c). Fewer participants in Acetazolamide and Combined groups developed symptoms (defined as AMS score ≥ 1) and eventually developed AMS. The proportion of subjects scoring three or higher was less in Combined group than in Acetazolamide group (6% vs 18%, P = 0.065). Regarding AMS symptoms, 107 (42.8%) participants experienced headaches, followed by 101 (40.4%) developed dizziness/light-headedness, 88 (35.2%) fatigue and/or weakness, and 26 (10.4%) gastrointestinal symptoms. Fewer participants in Acetazolamide and Combined groups suffered from headaches (Table 1).

SpO₂ was assessed in five groups after 6 h of hypoxic exposure (Fig. 4). Combined and Acetazolamide groups showed significantly ameliorated SpO₂ compared to the other groups, and the differences were detected as early as 1 h after being exposed to hypoxia. At 6 h, the SpO₂ in Control, Ripc, Rapid-Ripc, Acetazolamide, and Combined group were 85.82 ± 6.40%, 86.90 ± 4.46%, 83.80 ± 6.77%, 90.92 ± 3.77%, and 89.24 ± 4.17%, respectively. Significant differences were found between Combined and Acetazolamide with other groups (Acetazolamide vs Control: 5.06%, 95% CI 2.12 to 8.00, P < 0.001; vs Ripc: 4.04%, 95% CI 1.07 to 7.01, P < 0.001; vs Rapid-Ripc: 7.08%, 95% CI 4.13 to 10.02, P < 0.001; Combined vs Control: 3.42%, 95% CI 0.49 to 6.35, P = 0.020; vs Rapid-Ripc: 5.44%, 95% CI 2.51 to 8.37, P < 0.001).

Drug-related adverse reactions occurred in 44 (44.0%) participants taking acetazolamide, with paresthesia being the most commonly reported symptom, followed by polyuria and taste disturbance (Table 2). The majority of these reactions were classified as mild. There was no significant difference between Acetazolamide and Combined group.

The total number of adverse reactions in Ripc, Rapid-Ripc, Acetazolamide, and Combined group were 11 (22%), 20 (40%), 25 (50%), and 27 (54%), respectively. The incidence in Ripc group was lower than the other groups, and the differences were significant compared to Acetazolamide (P = 0.004) or Combined group (P = 0.001). The incidence of adverse effects did not significantly increase when Acetazolamide and Rapid-Ripc were used in combination (Combined group) compared to when Acetazolamide or Rapid-Ripc was used alone (P = 0.689 and P = 0.161, respectively). Taken together, Combined group had no significant increase in the incidence of adverse reactions.

The systolic and diastolic blood pressure and heart rate showed little fluctuation during the hypoxic exposure and did not exhibit significant difference between the groups. The results are showed in Additional file 1: Figure S2.