Metabolites of L-ARG in Exhaled Breath Condensate and Serum Are Not Biomarkers of Bronchial Asthma in Children
Abstract
:1. Introduction
2. Materials and Methods
2.1. Participants
2.2. Medical History and Physical Examination
2.3. Exhaled Breath Condensate Collection
2.4. Baseline Functional Respiratory Test
2.5. Atopy Status
2.6. Blood Sample Collection, Storage, and Preparation
2.7. Interleukin-4 Concentrations
2.8. L-ARG, ADMA, SDMA, CIT, ORN, and DMA Concentrations
- 100 µL aliquots of calibration standards or 100 µL of serum, 10 µL of internal standard solution (50 µM D6-DMA, 20 µM D7-ADMA, 100 µM D6-ornithine, and 100 µM D7-arginine, respectively), and 50 µL of borate buffer (0.025 M Na2B4O7 × 10H2O, 1.77 mM NaOH, pH = 9.2) were added into 2.0 mL polypropylene tubes and vortexed (1 min, 25 °C).
- Derivatization was performed using 400 µL of acetonitrile (ACN) and 10 µL of 10% BCl in ACN. The samples were incubated and vortexed (5 min, 25 °C), and centrifuged (7 min, 17,000 RCF, 4 °C). After this, 100 µL of the clear supernatant was transferred into glass tubes containing 400 µL of water.
- Preparation of standard calibration curves was performed using the following concentration ranges: 5; 12.5; 25; 50; 100; 150; 200; 250 µM for L-ARG, 0.05; 0.13; 0.25; 0.5; 1.0; 1.5; 2; 2.5 µM for ADMA and SDMA, 1; 2.5; 5; 10; 20; 30; to 50 µM for CIT, 2.5; 7.5; 15; 30; 60; 90; 120; 150 µM for ORN, and 0.14; 0.35; 0.7; 1.4; 2.8; 4.2; 5.6; 7.0 µM for DMA.
- Analytical chromatography was performed using an Acquity UPLC system containing a cooled autosampler (Waters, Milford, MA, USA) and Acquity HSS T3 column (50 × 1.0 mm, 1.75 µm) from Waters. Elution was conducted with 0.1% formic acid (FA) in water as mobile phase A and 0.1% FA in methanol as mobile phase B. The volume was 2 µL. Total run time was 7 min, with a total flow rate of 250 µL/min. The following gradient was used: 5% B for 0–0.5 min, 5–14% B for 0.5–3 min, 14–60% B for 3–4 min, 60–90% B for 4–4.5 min, 90% B for 4.5–5 min and 90–5% B for 5–5.10 min.
- Mass spectrometric analysis was conducted using Xevo G2 QTOF MS (Waters, Milford, MA, USA) with ESI in positive ion mode. Parameters such as the spray voltage, source temperature, and the desolvation temperature were set at 0.5 kV, 120 °C, and 450 °C, respectively. Nitrogen was used as the nebulizing and drying gas. Data were acquired by using MassLynx software (Waters, Milford, MA, USA) for the following ions (m/z): 279.1457 (for L-ARG), 286.1749 (for D7-arginine), 307.1717 (for ADMA and SDMA), 314.2076 (for D7-ADMA), 280.1297 (for CIT), 341.1501 (for ORN), 347.1878 (for D6-ornithine), 150.0919 (for DMA), and 156.1113 (for D6-DMA).
2.9. Statistical Analysis
3. Results
3.1. Characteristics of Participants
3.2. Biochemical Analyses
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Characteristic | Study Group n = 37 | Control Group n = 28 | p |
---|---|---|---|
Gender, n (%): | 0.547 | ||
Girls | 12 (32.4%) | 12 (42.9%) | |
Boys | 25 (67.6%) | 16 (57.1%) | |
Age (years): | 0.010 | ||
M ± SD | 10.9 ± 2.7 | 12.6 ± 2.5 | |
Me [Q1; Q3] | 10 [9; 13] | 13 [12; 14] | |
Min–Max | 6–17 | 7–17 | |
Age groups, n (%): | 0.002 | ||
6–11 years | 25 (67.6%) | 7 (25.0%) | |
12–17 years | 12 (32.4%) | 21 (75.0%) | |
Residential area: | 0.291 | ||
Rural | 10 (27.0%) | 12 (42.9%) | |
<15.000 residents | 4 (10.8%) | 1 (3.6%) | |
>15.000 residents | 23 (62.2%) | 15 (53.6%) | |
BMI (kg/m2): | 0.012 | ||
M ± SD | 18.6 ± 3.9 | 21.0 ± 3.4 | |
Me [Q1; Q3] | 17 [16; 21] | 21 [19; 23] | |
Min–Max | 12–29 | 15–30 | |
Number of siblings, n (%) | 0.429 | ||
M ± SD | 1.4 ± 1.1 | 1.3 ± 1.6 | |
Me [Q1; Q3] | 1 [1; 2] | 1 [1; 1] | |
Min–Max | 0–5 | 0–9 | |
Contact with an animal in the home environment: | |||
Dog | 12 (32.4%) | 10 (35.7%) | 0.990 |
Cat | 11 (29.7%) | 10 (35.7%) | 0.808 |
Exposure to tobacco smoke in the home | 13 (35.1%) | 12 (42.9%) | 0.707 |
Participation in physical education classes | 36 (97.3%) | 28 (100.0%) | 1.000 |
Pneumococcal vaccines | 16 (43.2%) | 7 (25.0%) | 0.207 |
FEV1 (% predicted) | 96.5 ± 15.0 | 104.8 ± 16.9 | 0.041 |
FVC (% predicted) | 107.8 ± 15.0 | 110.6 ± 14.3 | 0.441 |
FEV1/FVC | 89.3 ± 11.0 | 94.1 ± 10.2 | 0.075 |
Characteristic | Results |
---|---|
The age of diagnosis of asthma | |
M ± SD | 6.4 ± 3.6 |
Me [Q1; Q3] | 6 [4; 9] |
Min–Max | 1–13 |
Asthma duration | |
M ± SD | 4.6 ± 3.7 |
Me [Q1; Q3] | 4 [1; 7] |
Min–Max | 0.5–12 |
Diagnosis of asthma before 6 years of age | 17/37 (45.9%) |
Other atopic diseases in the child | 23/37 (62.2%) |
Positive family history of atopic diseases | 23/37 (62.2%) |
Atopy | 27/37 (73.0%) |
Treatment with inhaled corticosteroids in the previous 4 weeks | 30/37 (81.1%) |
FEV 1%FVC (% predicted) | |
M ± SD | 89.3 ± 11.0 |
Me [Q1; Q3] | 92 [83; 95] |
Min–Max | 64–116 |
FEV 1 (% predicted) | |
M ± SD | 96.5 ± 15.0 |
Me [Q1; Q3] | 97 [91; 105] |
Min–Max | 57–130 |
FVC (% predicted) | |
M ± SD | 107.8 ± 15.0 |
Me [Q1; Q3] | 108 [96; 113] |
Min–Max | 85–152 |
Serum L-ARG and Its Metabolite Concentration | Study Group n = 36 | Control Group n = 26 | p |
---|---|---|---|
L-ARG (μmol/L) | 0.881 | ||
M ± SD | 118.16 ± 28.32 | 121.81 ± 48.52 | |
Me [Q1; Q3] | 115.0 [96.4; 132.7] | 122.7 [90.1; 139.1] | |
Min–Max | 76.7–220.9 | 45.3–290.7 | |
ADMA (μmol/L) | 0.304 | ||
M ± SD | 0.56 ± 0.11 | 0.54 ± 0.18 | |
Me [Q1; Q3] | 0.55 [0.50; 0.60] | 0.52 [0.44; 0.60] | |
Min–Max | 0.35–0.81 | 0.28–1.18 | |
SDMA (μmol/L) | 0.416 | ||
M ± SD | 0.30 ± 0.07 | 0.32 ± 0.10 | |
Me [Q1; Q3] | 0.29 [0.25; 0.36] | 0.31 [0.27; 0.35] | |
Min–Max | 0.17–0.47 | 0.17–0.70 | |
CIT (μmol/L) | 0.653 | ||
M ± SD | 29.59 ± 8.69 | 28.45 ± 7.83 | |
Me [Q1; Q3] | 28.1 [24.7; 33.5] | 27.5 [24.3; 33.1] | |
Min–Max | 12.3–54.3 | 16.0–48.9 | |
ORN (μmol/L) | 0.972 | ||
M ± SD | 52.87 ± 20.38 | 63.40 ± 52.35 | |
Me [Q1; Q3] | 45.5 [37.0; 70.0] | 50.8 [38.0; 58.7] | |
Min–Max | 27.6–111.9 | 16.7–242.7 | |
DMA (μmol/L) | 0.858 | ||
M ± SD | 1.62 ± 0.67 | 1.67 ± 0.73 | |
Me [Q1; Q3] | 1.6 [1.1; 2.1] | 1.6 [1.0; 2.1] | |
Min–Max | 0.5–3.5 | 0.7–3.8 | |
ADMA/L-ARG ratio (%) | 0.112 | ||
M ± SD | 0.48 ± 0.12 | 0.49 ± 0.29 | |
Me [Q1; Q3] | 0.5 [0.4; 0.5] | 0.4 [0.4; 0.5] | |
Min–Max | 0.3–0.9 | 0.3–1.7 | |
SDMA/L-ARG ratio (%) | 0.633 | ||
M ± SD | 0.26 ± 0.07 | 0.29 ± 0.15 | |
Me [Q1; Q3] | 0.3 [0.2; 0.3] | 0.3 [0.2; 0.3] | |
Min–Max | 0.1–0.4 | 0.2–1.0 | |
DMA/L-ARG ratio (%) | 0.960 | ||
M ± SD | 1.40 ± 0.57 | 1.54 ± 1.11 | |
Me [Q1; Q3] | 1.3 [1.0; 1.7] | 1.3 [1.1; 1.6] | |
Min–Max | 0.5–2.7 | 0.6–6.5 |
Serum IL-4 Concentration | Study Group n = 37 | Control Group n = 27 | p |
---|---|---|---|
IL-4 (pg/mL) | 0.716 | ||
M ± SD | 0.16 ± 0.05 | 0.15 ± 0.04 | |
Me [Q1; Q3] | 0.14 [0.13; 0.17] | 0.14 [0.12; 0.16] | |
Min–Max | 0.10–0.34 | 0.11–0.28 |
L-ARG and Its Metabolite Concentrations in EBC | Study Group n = 32 | Control Group n = 20 | p |
---|---|---|---|
L-ARG (μmol/L) | 0.672 | ||
M ± SD | 17.01 ± 29.38 | 16.48 ± 29.23 | |
Me [Q1; Q3] | 5.9 [3,7; 10.6] | 5.9 [4.5; 13.3] | |
Min–Max | 1.7–121.5 | 1.5–127.9 | |
ADMA (μmol/L) | 0.323 | ||
M ± SD | 0.31 ± 0.42 | 0.29 ± 0.40 | |
Me [Q1; Q3] | 0.2 [0.2; 0.2] | 0.2 [0.2; 0.2] | |
Min–Max | 0.1–1.9 | 0.1–1.9 | |
SDMA (μmol/L) | 0.352 | ||
M ± SD | 0.16 ± 0.08 | 0.14 ± 0.05 | |
Me [Q1; Q3] | 0.1 [0.1; 0.1] | 0.1 [0.1; 0.1] | |
Min–Max | 0.1–0.4 | 0.1–0.3 | |
CIT (μmol/L) | 0.125 | ||
M ± SD | 27.39 ± 34.99 | 37.77 ± 70.70 | |
Me [Q1; Q3] | 15.9 [13.4; 20.3] | 18.0 [16.0; 25.7] | |
Min–Max | 10.8–159.3 | 10.9–332.7 | |
ORN (μmol/L) | 0.108 | ||
M ± SD | 40.23 ± 65.02 | 80.73 ± 212.05 | |
Me [Q1; Q3] | 15.7 [11.1; 29.5] | 28.4 [15.3; 38.3] | |
Min–Max | 3.8–295.2 | 7.8–967.6 | |
DMA (μmol/L) | 0.211 | ||
M ± SD | 14.46 ± 6.48 | 17.13 ± 7.28 | |
Me [Q1; Q3] | 14.2 [9.2; 16.8] | 15.2 [11.6; 21.5] | |
Min–Max | 4.8–33.9 | 5.8–34.7 | |
ADMA/L-ARG ratio (%) | 0.579 | ||
M ± SD | 3.31 ± 1.80 | 3.17 ± 2.23 | |
Me [Q1; Q3] | 3.0 [1.7; 4.3] | 2.8 [1.6; 3.9] | |
Min–Max | 0.7–8.5 | 1.1–9.8 | |
SDMA/L-ARG ratio (%) | 0.592 | ||
M ± SD | 2.49 ± 1.68 | 2.32 ± 1.92 | |
Me [Q1; Q3] | 2.2 [1.2; 3.5] | 2.2 [1.0; 2.9] | |
Min–Max | 0.3–7.4 | 0.3–8.3 | |
DMA/L-ARG ratio (%) | 0.843 | ||
M ± SD | 274.29 ± 219.47 | 290.41 ± 231.87 | |
Me [Q1; Q3] | 219.3 [112.3; 428.9] | 203.2 [110.5; 420.4] | |
Min–Max | 8.1–764.7 | 15.6–795.0 |
Parameter | L-ARG | ADMA | SDMA | CIT | ORN | DMA | ADMA/ L-ARG | SDMA/ L-ARG | DMA/ L-ARG |
---|---|---|---|---|---|---|---|---|---|
FEV1 and serum (n = 62) measurements | 0.076 | 0.142 | 0.175 | 0.110 | −0.022 | 0.109 | −0.064 | 0.048 | 0.045 |
FEV1 and EBC (n = 52) measurements | −0.090 | −0.151 | −0.040 | −0.202 | −0.161 | 0.012 | 0.011 | 0.077 | 0.071 |
IL-4 and serum (n = 62) measurements | −0.014 | 0.039 | 0.109 | 0.213 | 0.160 | 0.080 | 0.065 | 0.082 | 0.136 |
IL-4 and EBC (n = 51) measurements | −0.050 | −0.014 | 0.018 | −0.053 | 0.080 | 0.102 | 0.064 | 0.038 | 0.088 |
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Połomska, J.; Sozańska, B. Metabolites of L-ARG in Exhaled Breath Condensate and Serum Are Not Biomarkers of Bronchial Asthma in Children. J. Clin. Med. 2022, 11, 252. https://doi.org/10.3390/jcm11010252
Połomska J, Sozańska B. Metabolites of L-ARG in Exhaled Breath Condensate and Serum Are Not Biomarkers of Bronchial Asthma in Children. Journal of Clinical Medicine. 2022; 11(1):252. https://doi.org/10.3390/jcm11010252
Chicago/Turabian StylePołomska, Joanna, and Barbara Sozańska. 2022. "Metabolites of L-ARG in Exhaled Breath Condensate and Serum Are Not Biomarkers of Bronchial Asthma in Children" Journal of Clinical Medicine 11, no. 1: 252. https://doi.org/10.3390/jcm11010252
APA StylePołomska, J., & Sozańska, B. (2022). Metabolites of L-ARG in Exhaled Breath Condensate and Serum Are Not Biomarkers of Bronchial Asthma in Children. Journal of Clinical Medicine, 11(1), 252. https://doi.org/10.3390/jcm11010252