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ACS omega2025; 10(41); 48412-48417; doi: 10.1021/acsomega.5c05941

Intraday and Interday Evaluation of pH and Hydrogen Peroxide in the Exhaled Breath Condensate of Horses Using A Portable Device.

Abstract: The analysis of equine exhaled breath condensate (EBC) lacks standardized methodology, and current collection devices are often adapted for research. This study evaluates a novel horse-specific EBC collector and assesses the variability of EBC pH and hydrogen peroxide (HO) levels, exploring potential correlations with bronchoalveolar lavage (BAL) and tracheal wash (TW) cytology. Eleven healthy mixed-breed mares from a teaching herd, with no evidence of airway abnormalities, were included in this randomized observational study. The collection efficiency of the proposed device was assessed, and intra- and interday variations in EBC pH and HO levels were analyzed. Airway endoscopy, tracheal mucus scoring, and TW and BAL fluid cytology were also performed. EBC pH showed no significant intra- ( = 0.631, ES 0.008-0.456) or interday ( = 0.864, ES 0.116-0.365) variation, nor did HO levels ( = 0.953, ES 0.077-0.185; = 0.929, ES 0.019-0.190, respectively). In this study, no correlations were found between EBC parameters and BAL or TW cytology. However, 34.5% of pH samples and 32.7% of HO samples were insufficient for analysis due to low sample volume. These findings suggest that EBC collection using the horse-specific device is feasible and that pH and HO levels remain stable regardless of collection time. However, further refinement of the device is necessary to improve sample yield and ensure reliable analysis.
© 2025 The Authors. Published by American Chemical Society.
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Publication Date: 2025-10-06 PubMed ID: 41141810PubMed Central: PMC12547516DOI: 10.1021/acsomega.5c05941Google Scholar: Lookup
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Summary

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Overview

  • This study evaluated a new horse-specific device designed to collect exhaled breath condensate (EBC) from horses and measured the stability of pH and hydrogen peroxide levels in these samples over time.
  • The research also explored whether these EBC measurements correlated with respiratory health indicators from bronchoalveolar lavage (BAL) and tracheal wash (TW) cytology, finding no strong correlations but demonstrating the device’s general feasibility.

Background and Objective

  • Exhaled breath condensate (EBC) is a non-invasive method to sample airway secretions and can be analyzed for biomarkers such as pH and hydrogen peroxide (HO), which may reflect respiratory health.
  • In horses, standardized methods for collecting and analyzing EBC are lacking, and most existing collection devices are adapted from other species or designed primarily for research purposes rather than routine clinical use.
  • The main goals of this study were to:
    • Evaluate a novel, horse-specific EBC collection device for efficiency and usability.
    • Assess intraday (within the same day) and interday (across different days) variability in EBC pH and HO measurements to determine their stability over time.
    • Examine possible correlations between EBC measurements and established respiratory health indicators (cytological analysis of BAL and TW fluids).

Methods

  • The study involved eleven healthy mixed-breed mares from a teaching herd, confirmed to have no respiratory abnormalities through clinical exam and airway evaluation.
  • Key procedures included:
    • Collection of EBC samples using the new, horse-specific portable device.
    • Measurement of pH and hydrogen peroxide concentrations in EBC samples.
    • Evaluation of intraday and interday variability by repeated sampling on the same and different days.
    • Respiratory health assessment by airway endoscopy, scoring of tracheal mucus, and cytological analysis of samples obtained from bronchoalveolar lavage (BAL) and tracheal wash (TW).

Key Findings

  • The new collection device was capable of sampling EBC from horses, but around one-third of the collected samples for both pH (34.5%) and hydrogen peroxide (32.7%) were insufficient in volume for reliable analysis, indicating the need for further device improvement.
  • Statistical analysis showed no significant intraday or interday variation in EBC pH and HO levels:
    • pH intra-day variability: p=0.631, effect size (ES) 0.008–0.456
    • pH inter-day variability: p=0.864, ES 0.116–0.365
    • HO intra-day variability: p=0.953, ES 0.077–0.185
    • HO inter-day variability: p=0.929, ES 0.019–0.190
  • No statistically significant correlations were found between EBC parameters (pH and HO levels) and the cytological findings from BAL and TW samples, suggesting that EBC markers in this healthy population may not reflect subtle airway inflammation detectable by these standard methods.

Implications and Conclusions

  • The study demonstrated that the novel, horse-specific EBC collection device can be used to obtain pH and hydrogen peroxide measurements in equine breath condensate with repeatability over time.
  • Stability of pH and HO levels over hours and days suggests these biomarkers are consistent and potentially reliable metrics when collected properly.
  • However, the relatively high rate of insufficient sample volume points to a need for optimizing the device design to improve condensate collection efficiency.
  • The lack of correlation with BAL and TW cytology in this healthy cohort suggests that further research in horses with respiratory disease is needed to evaluate whether these EBC biomarkers can serve as meaningful clinical indicators.
  • Overall, this work contributes a useful tool and normative data for future development of non-invasive respiratory diagnostics in horses.

Cite This Article

APA
Barbosa B, Santi TF, Rodak AC, Nogara MF, Leite LMB, Weber SH, Niels C, Daros RR, Michelotto PV. (2025). Intraday and Interday Evaluation of pH and Hydrogen Peroxide in the Exhaled Breath Condensate of Horses Using A Portable Device. ACS Omega, 10(41), 48412-48417. https://doi.org/10.1021/acsomega.5c05941

Publication

ACS omega
ISSN: 2470-1343
NlmUniqueID: 101691658
Country: United States
Language: English
Volume: 10
Issue: 41
Pages: 48412-48417

Researcher Affiliations

Barbosa, Bianca
  • Graduate Program in Animal Science, Pontifícia Universidade Católica Paraná, Rua Imaculada Conceição 1155, Prado Velho, 80215-901 Curitiba, PR, Brazil.
Santi, Thasla F
  • Graduate Program in Animal Science, Pontifícia Universidade Católica Paraná, Rua Imaculada Conceição 1155, Prado Velho, 80215-901 Curitiba, PR, Brazil.
Rodak, Ana C
  • Veterinary Medicine, Pontifícia Universidade Católica Paraná, Rua Imaculada Conceição 1155, Prado Velho, 80215-901 Curitiba, PR, Brazil.
Nogara, Maria F
  • Veterinary Medicine, Pontifícia Universidade Católica Paraná, Rua Imaculada Conceição 1155, Prado Velho, 80215-901 Curitiba, PR, Brazil.
Leite, Lidiane M B
  • Core for Cell Technology, School of Medicine and Life Sciences, Pontifícia Universidade Católica do Paraná, Rua Imaculada Conceição 1155, Prado Velho, 80215-901 Curitiba, PR, Brazil.
Weber, Saulo H
  • Graduate Program in Animal Science, Pontifícia Universidade Católica Paraná, Rua Imaculada Conceição 1155, Prado Velho, 80215-901 Curitiba, PR, Brazil.
Niels, Cleber
  • Graduate Program in Animal Science, Pontifícia Universidade Católica Paraná, Rua Imaculada Conceição 1155, Prado Velho, 80215-901 Curitiba, PR, Brazil.
Daros, Ruan R
  • Graduate Program in Animal Science, Pontifícia Universidade Católica Paraná, Rua Imaculada Conceição 1155, Prado Velho, 80215-901 Curitiba, PR, Brazil.
  • EthoLab - Applied Ethology and Animal Welfare Lab, Pontifícia Universidade Católica do Paraná, Rua Imaculada Conceição 1155, Prado Velho, 80215-901 Curitiba, PR, Brazil.
Michelotto, Pedro V
  • Graduate Program in Animal Science, Pontifícia Universidade Católica Paraná, Rua Imaculada Conceição 1155, Prado Velho, 80215-901 Curitiba, PR, Brazil.
  • EthoLab - Applied Ethology and Animal Welfare Lab, Pontifícia Universidade Católica do Paraná, Rua Imaculada Conceição 1155, Prado Velho, 80215-901 Curitiba, PR, Brazil.

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