Online Supplement
Response-dose ratio is a surrogate of cumulative provocative dosage for bronchial provocation
tests in asthma
Wei-jie Guan, Ph. D.; Jin-ping Zheng, M. D.; Xu Shi, Ph. D.; Yan-qing Xie, Ph. D.; Cai-yu Jiang *, M.
Med.; Yi Gao, M. Med.; Jia-ying An, M. T.; Xin-xin Yu, M. T.; Wen-ting Liu, M. T.
State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory
Disease, First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China
* Sichuan People’s Hospital, Chengdu, Sichuan, China
Corresponding author: Jin-ping Zheng, M. D., Address: 151 Yanjiang Road, Guangzhou, Guangdong,
China, Fax: +86-20-83062729, Phone: +86-20-83062869, E-mail: jpzhenggy@163.com
Conception and design: J. Z., W. G., Y. G. Y. X.; Analysis and interpretation: W. G., C. J., X. S., Q. L., Z.
Z., E. G., J. A., X. Y., W. L.; Drafting the manuscript for important intellectual content: W. G., J. Z., N.
Z.
Running title: RDR and PD20FEV1 in asthma
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Preparations of LTD4 and MCh dilutions
The methods for preparing LTD4 dilutions have been described previously【1,2】. Briefly, LTD4
(200 nmol/ml in ethanol; Cayman Chemical Co., Ann Arbor, Mich) was stored in a -80℃ freezer.
The 0.2ml LTD4 aliquot was extracted using a micropipette. And the 32 μmol/L LTD4 dilution
(solution A) was prepared by adding 1.050 ml normal saline. The aliquot of solution A was then
pipetted into 0.3 ml normal saline forming 16 μmol/L LTD4 dilution (solution B). Through similar
way was ethanol dilution control (16% ethanol) prepared, except that 0.20ml ethanol absolute
was adopted.
Methacholine (Sigma-Aldrich Co, St Louis, Mo) was stored in a -20℃ freezer. A gram of
methacholine was solved using 10ml normal saline, the dilution control, for preparation of 5.0×105
μmol/L dilution (solution C). The 2.5×105 μmol/L dilution (solution D) was prepared by adding an
aliquot of normal saline into 3ml solution C.
Reference
1. Guan W, Zheng J, Gao Y, Jiang C, Xie Y, An J, Yu X, Liu W, Zhong N. Leukotriene D4 and
methacholine bronchial provocation test for identifying leukotriene-responsiveness subtypes. J
Allergy Clin Immunol. 2013, 131: 332-8.
2. Guan W, Zheng J, Gao Y, Jiang CY, An J, Yu X, Liu W. Leukotriene D4 bronchial provocation test:
methodology and diagnostic value. Curr Med Res Opin. 2012, 28: 797-803.
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Table E1
Definition of asthma control
Controlled
Partly controlled
(All of the following)
(Any measure present)
Daytime symptoms
None (twice or less/week)
More than twice/week
Limitation of activities
None
Any
None
Any
Uncontrolled
Nocturnal symptoms/
awakening
Three or more features
Need for reliever/rescue
of partly controlled
None (twice or less/week)
More than twice/week
treatment
＜80% predicted or
Lung function
Normal
(FEV1 or PEF)
personal best (if known)
* Criteria were defined based on GINA updated 2009.
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Table E2
Stimuli
LTD4
Methacholine
Procedures of LTD4-BPT and MCh-BPT
Concentration
Inhalation
Inhalation
Dosage
Cumulative
(μmol/l)
times
duration (s)
(μmol)
dosage (μmol)
0
0
5
0.375
0.000
0
1
16
1
0.315
0.075
7.5×10-5
2
16
3
0.315
0.225
3.0×10-4
3
32
5
0.375
0.900
1.2×10-3
4
32
10
0.750
3.600
4.8×10-3
0
0
5
0.600
0.000
0.000
1
2.5×105
1
0.585
0.39
0.039
2
2.5×105
3
0.585
1.23
1.56
3
5.0×105
7
0.521
4.69
6.25
4
5.0×105
9
0.504
6.25
12.50
Step
For LTD4, 1 nanomole equals to 0.5 microgram; For MCh, 1 micromole is equivalent to 200 micrograms.
Both BPTs consisted of a single step of dilution inhalation challenge, and if warranted, 4 successive steps
with different inhalation times of stimuli, inhalation duration and concentrations. We have previously validated
the methodologies, which target at deriving the cumulative provocative dosage (PD), but not cumulative
provocative concentration (PC), despite that they differed from that stated in relevant guidelines
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