Methacholine R. G. Townley, F. D. Brodkey,
inhalation
challenge
studies
M.D., A. K. Bewtra, M.D., N. M. Nair, M.D., M.D., G. D. Watt, B.A., and K. M. Burke, B.S.
Omclhu, Neh.
Methacholine sensitivity has become a valuable and widely used technique for studying the irritability of the airways. Asthmatics are lOO- to l,OOO-fold more sensitive than normal subjects to various mediators such as methacholine (P-acetyl methacholine).‘-” This degree of sensitivity has been used to define asthma and also as a genetic marker.l The methacholine responsiveness may be determined by 1 of 2 methods: (1) by determining dose-response curves to increasing concentrations of methacholine while keeping the number of breaths and the volume of methacholine inhaled constant” and (2) by determining dose-response curves by keeping the concentration constant while increasing the number of inhalations of methacholine.” The first method is currently being used more widely and has been recommended by the American Academy of Allergy to provide a standard and uniform method. It is described in detail elsewhere.” The second method has been used since 1962 and has been the basis for a number of short-term and long-term studies. We have recently compared both of these methods to determine the short-term reliability of each method. The dose-response curves and thus the degree of bronchial sensitivity were determined in 19 subjects in a randomized 4-way crossover study. Each subject was challenged twice by each method at 1-wk intervals. The short-term reproducibility for both methods was good (r = 0.934 and 0.942). The correlation between methods was also significant (r =0.953). The various pulmonary function parameters that can be evaluated during an inhalation challenge are numerous and include the FVC, FEV,, SG,,,, FEF,,-,,, PEFR, flow volume loops, etc. The easiest and the most widely used currently is the FEV,, which is the minimum requirement for comparison of responses as recommended by the Standardization Committee, j From the Allergic Diseases Center, Department of Medicine, Creighton University School of Medicine. Supported in part by Grants 5P50 AI12029 and l-HR-2-2985 from the National Institutes of Health. Reprint requests to: R. G. Townley, M.D., Creighton University School of Medicine, Omaha, NE 68179. 0091-6749/79/130569+06$00.60/O
@I 1979 The C. V. Mosby
Special abbreviations used FVC: forced vital capacity FEV,: forced expiratory volume at I set SG,,.: specific airway conductance FEF25-73: PEFR:
forced expiratory flow at 25% to 75% peak expiratory flow rate
A drop in FEV, of 20% or greater from the baseline with any of the standard concentrations of methacholine is considered a positive test. As seen in Table I, we have subcategorized the degree of responsiveness with respect to dose of methacholine (inhalations) and pulmonary function (percent baseline FEV,). During the course of these studies, we found a subgroup of allergic rhinitis patients who had a decrease of 20% or greater in FEV, but who plateaued out later with increasing doses of methacholine,‘. L i as shown in Fig. 1. Some of these patient? actually showed an improvement in their pulmonary function tests with higher doses of methacholine. According to our criteria, they would have been classified as having high- or medium-positive reactions. To avoid this false high-positive classification, we found in our studies that the area under the dose-response curve is more reliable in defining the degree of sensitivity.“,
i, x
Area is calculated by integrating the best-fit parabola of the methacholine dose-response curve (Fig. 2). The test cutoff point is either a 35% drop in FEV, or 160 inhalations of methacholine. Area values that correspond to the previously defined response categories are shown in Table I. Analysis of our data in over 1,500 methacholine challenges shows the following facts: 1. Sex of the individual has no effect on the methacholine responsiveness. 2. Age does affect the methacholine responsiveness. Children and older individuals are more sensitive, and at least for research purposes, all values should be age corrected. 3. In a large group of patients, correlation of methacholine sensitivity with the histamine sensitivity is statistically significant; however, in a given individual Co.
Vol. 64, No. 6, Part 2, pp. 569-574
570
Townley
et al.
J ALLERGY
CLIN. IMMUNOL DECEMBER 1979
DEGREES OF METHACHOLINE RESPONSE
01 0
, 1020
40
METHACHOLINE
60 INHALATIONS
FIG. 1. Rapidly decreasing dose-response curves for 2 shows no change during the course of the challenge. somewhat shallower curve, which eventually exceeds strates the plateau phenomenon. His initial response 20%; however, additional inhalations did not cause a TABLE I. Characterization
Category
High Medium Low Negative
of methacholine
% Drop in FEV,
2 20% 220% 220% -=200/c
inhalation
Total no. of breaths 0.1-10
II-40 41-160 160
1 160
(5mg/ml)
asthmatics (46-Z 39-11). A normal (47-71 Subject 47-5 is an asthmatic who has a a 35% drop in FEV,. Subject 5-4 demonat 20 and 40 inhalations is greater than further drop in pulmonary function.
responses Ratio =
% Drop in FEV, No. of breaths z2.00
0.50-1.99 0.13-0.49 SO.125
Area under the curve O-306.25
306.25-1,225 1,225-4,000 4,000-5,600
Legend: We consider persons with areas of less than 306 to be high-positive responders. Areas from 306 to 1,225 are classed as medium-positive; 1,225 to 4,000 as low-positive; and 4,000 to 5,600 as negative. These classes were obtained from traditional singlepoint estimates of response, i.e., High = fall in FEVl greater than 20% after 10 cumulative inhalations of methacholine aerosol at 5 mgiml; Medium = greater than 20% after 40 cumulative inhalations; Low = greater than 20% after 160 cumulative inhalations; and Negative = less than 20% fall after 160 cumulative inhalations.
this may not be true.l. 6, 9 A person who is highly sensitive to one mediator may be less sensitive to the other and vice versa. 4. We have never seen a reaction severe enough to require hospitalizing a patient. Most patients reversed to normal pulmonary functions within 5 min following bronchodilators like isoproterenol or within 30 to 45 rnin without any bronchodilator. 5. We saw no incidences of delayed reactions to methacholine. 6. The test is reproducible and reliable over both short-term and long-term follow-up studies. To evaluate the familial nature of asthma and the methacholine responsiveness, we studied 47 atopic and 26 nonatopic families. Atopic families had at
patient,
least 1 child with current asthma. There were 648 members in these families. In 26 nonatopic families 237 members had no reported history of asthma, hay fever, or atopic eczema for at least 3 generations of first- and second-degree relatives. The proband, his or her siblings, parents, and grandparents made up the study group. All probands were excluded for genetic analysis. From these studies of methacholine responsiveness (Fig. 3), we have shown that: 1. Over 90% of asthmatics have high- or medium-positive responsiveness to methacholine. Only 3 individuals’ responses were negative, and they are former asthmatics who have been completely free of symptoms for several years. Most asthmatics who
VOLUME NUMBER
Methacholine
64
inhalation
challenge
studies
571
6, PART 2
METHACHOLINE~5mghl)
CHALLENGE
70 5 L? 50 (3
0
ALLERGIC
q q
NORMAL(ATOPIC
RHlNlTls FAMILIES)
NORMAL(NON-ATOPIC
FAMILIES)
O” 30 $ 8
a
10
aw HIGH
MEDIUM
CUMULATIVE
DOSE
as defined
NEGATIVE
RESPONSE
METHACHOLlNE
%““1 *,,: $y+bx+c;rh:ra&: Pysqr16aFIG. 3. Response IO 40 4 160 ,I 4 I
LOW
in Table I; see text for details.
(INHALATIONS)
FIG. 2. A least-squares fitted, parabolic dose-response curve. Provocative dose at 35% (PDz5) is the right-hand limit (x,,) of the integral. The X axis has been logarithmically transformed for ease of presentation.
cease to have attacks remain methacholine positive for several years after their last attack, though the degree of their sensitivity is only ‘/IO that of current asthmatics.l. 9. 1 2. Less than 5% of individuals with hay fever or nonatopic normal subjects (normals) show a highpositive response. 3. Twenty-seven percent of hay fever patients had a negative response compared to 49% of normals. 4. Hay fever patients and normals had about the same incidence of low-positive responses. 5. Thirty percent of hay fever patients had a medium-positive response compared to 18% of normals from families with a history of asthma and 8% of normals from control families. 6. The normals from atopic families had significantly fewer negative and more positive responses than normals from normal families. Chi-square analysis of our data showed that: 1. Normals from asthma families were significantly different from normals from control families (p < 0.01). 2. Hay fever patients were not significantly different from normals from atopic families (p > 0.05). 3. Asthmatics were different from all other groups. 4. Hay fever patients were different from normals of normal families only. When the frequency of positive responses for the methacholine challenge in the rzormuls from atopic and nonatopic families was determined, 2 peaks or subpopulations were obtained in the atopic (asthmatic) families (Fig. 4). In contrast, the normals from nonatopic families
0 -200
1080
Methacholine
3640
2360
5600
Area
FIG. 4. This is a frequency plot of age-corrected area under the dose-response curves for methacholine in normals from both asthma and control families. The lefthand mode shows the excess number of positive responders from asthma families.
have a single uniform distribution with no corresponding subpopulation of the methacholine responders. The individuals with a peak in the positive test may be potential asthmatics, and this bimodal distribution in the atopic families suggests a single biochemical or physical defect in asthma. The accuracy and usefulness (i.e., sensitivity and specificity) of the methacholine response test have also been evaluated (Fig. 5). Sensitivity is defined as the ratio of the true positives to the sum of the true positives plus false negatives for the whole population. Specificity is defined as the ratio of the true negatives and the sum of the true negatives plus false positives in the nonatopic population. As can be seen, as the area under the dose-response curve cutoff value
572
Townley
J ALLERGY
et al.
c---r
SENSITIVITY
("HOLE POPULATION)
e--o
SPtcIFICITY
TRUE NEGATIVES
(NON-ATOPIC FAMILIES)
I 0
1 1700
900
Methacholine
Area
-
TRUE NEGATIVES + FALSE Pos,T,"ES
I.
Cut
FIG. 5. Comparing response to methacholine with clinical 1,300 optimized both sensitivity and specificity at 90%.
‘“lC’I,&CH?L,NF
HISTAMINC
MFTHACHOLIW
20% FEV, PERCENT
H,STAMhE
35% DROP
FIG. 6. A graph of the mean (+ SEM) provocative doses of methacholine and histamine for 20% and 35% falls (PD2,, and PD,,) following placebo, SCH 1000, and metaproterenol.
increases, the sensitivity continues to increase and specificity continues to decrease. The best sensitivity and specificity were obtained at an area of 1,300; however, the range 800 to 2,000 was acceptable. Since the low-positive responses range from 1,225 to 4,000, one has to be very careful in interpreting results beyond 2,000 for fear of falsely labeling them positive. Figs. 6 and 7 show the results of the effects of 40 pg 8-isopropylnoratropine methabromide (SCH 1000) or atrovent or 1.3 mg metaproterenol or placebo on methacholine and histamine challenges in 10 subjects with mild-to-moderate asthma. These challenges were performed in a double-blind and randomized fashion. This study was done using method
‘b
TRUE POSITIW .~~....~~ ~.~ ~~~~~...~~~. TRUE POSITIVFS + FALSE NEGATIVES
=
CLIN. IMMUNOL DECEMBER 1979
8. 2500
Off
1
-
3300
-
I
'
Values
diagnosis
we found
that an area of
No. 1, i.e., increasing concentrations of methacholine and histamine administered via a dosimeter.” The results in Fig. 6 show that: 1. Methacholine at 1.31 + 1.0 and 8.6 + 7.4 mg was required to produce 20% and 35% decreases in FEV, , respectively. In the presence of SCH 1000, 30.3 t 7.9 (p < 0.01) and 47.9 t 6.5 mg (p < 0.001) and, in the presence of metaproterenol, 17.6 +- 6.3 (p < 0.02) and 32.3 2 8.9 (p < 0.05) mg methacholine produced the same responses, respectively. 2. Histamine at 3.8 -+ 2.2 and 13.7 ? 6.8 mg was required to produce 20% and 35% drops in FEV,. In the presence of SCH 1000, 5.9 t 2.9 and 16.0 -+ 7.2 mg (p is not significant) and, in the presence of metaproterenol, 15.5 ? 4.2 (p < 0.02) and 33.9 & 8.0 (p = 0.05) mg histamine produced the same responses, respectively. 3. SCH 1000, an anticholinergic compound, antagonizes methacholine responses specifically while metaproterenol, a /3-adrenergic agonist, antagonizes both the histamine and methacholine bronchoconstriction. These studies indicate that the histamine bronchoconstriction in asthmatics is not due to a vagal reflex, as SCH 1000 should then have blocked the effects of both methacholine and histamine. DISCUSSION From our experience of performing over 1,500 methacholine challenges in patients with bronchial asthma since 1962 and in an additional 500 nonasthmatic persons, either atopic or nonatopic, we think methacholine sensitivity should be part of the definition of asthma. We have not observed a single nega-
VOLUME NUMBER
64 6, PART 2
tive inhalation challenge to methacholine in a patient who currently has bonafide bronchial asthma. ‘. A. ’ Among current asthmatics, the severity of asthma determines the bronchial sensitivity of subjects to methacholine. This sensitivity varies from 100 to several thousand times that of normal subjects .2. 6*“I- ” However, in former asthmatics, the degree of bronchoconstriction to methacholine was also related to the severity of past asthma symptoms.‘. “L I2 The mean sensitivity of former asthmatics is approximately ‘/IO that of current asthmatics. In a number of studies. the airway response to inhaled histamine has been found to be similar to that after methacholine.g, 12. I:‘. I.‘. I6 Those patients who are extremely sensitive to methacholine are also sensitive to histamine, although Spector and Farr” do report that a few patients sensitive to methacholine are only slightly sensitive to histamine and vice versa. Histamine is much more effective in producing bronchoconstriction when administered by aerosol than by the intravenous route, even though the intravenous route produces many more systemic responses such as flushing and throbbing headaches.” Many stimuli appear to cause reflex bronchoconstriction in asthmatic subjects in concentrations that evoke little or no response in healthy subjects. In man, Yu et al.‘# reported that atropine by intravenous or aerosol route markedly inhibits the bronchial constrictor response to inhaled antigen in asthmatic patients. Atropine and some recent atropine derivatives such as SCH 1000 (atrovent) are potent bronchodilators in many patients with asthma.lg Our results (Fig. 7) show SCH 1000 to result in some bronchodilation, but this was somewhat less than that produced by metaproterenol. Although SCH 1000 has been found to markedly inhibit the bronchoconstriction induced by methacholine, it has little or no effect in inhibiting histamine-induced bronchoconstriction. Thus our findings do not support the concept that histamine sensitivity is due to a heightened vagal reflex. In contrast, metaproterenol showed comparable protect’on against both methacholine and histamine. This suggests that these mediators are able to cause bronchoconstriction because they are not opposed by the intact P-adrenergic receptors that normally serve to maintain bronchodilatation. Subjects with allergic rhinitis and atopic eczema, as well as other members of asthmatic families, are more likely IO develop asthma.‘, i, “‘3 2’ However, which of these individuals will eventually develop asthma is not known. Those subjects with allergic rhinitis and atopic eczema or who are siblings of asthmatics and who have a high or medium response to methacholine
Methacholine
inhalation
challenge
studies
MEAN DOSE-RESPONSE HISTAMINE (n -I _ _ _ _M~T_APROTERENOL( -- -.__
573
CURVES =s)
1.3mQ) a,
\
\
i .
I I i+
g
I
METHACHOLINE C-..--t.jETAPROTERENOL -. ---__ 105-c SCH .-.__lOOo(40~~~ _ ----.
(n
ES)
(I.3mg) - *.., &
*--. --..
11
1
I
* -I:, ‘\
‘11 .i
65 t ‘POST DRUG BASELINE
’ I
I
I
h 4
CUMULATIV: DOSE30F CHALLENGE AGENTflog Hg) FIG. 7. Mean dose-response curves for histamine and methacholine challenges show the effects of pretreatment wih placebo, SCH 1000, and metaproterenol.
may be the ones at greater risk. By periodically studying these high-risk subjects before and after the pollen seasons and after respiratory infections, it may be possible to relate their methacholine sensitivity to the onset of asthma. Our results show that 30% of allergic rhinitis patients who were family members of an asthma family and 18% of nonatopic members of these same families had a medium-positive response to methacholine. This group, plus the 5% of hay fever subjects with a high-positive methacholine response represent the population who may be at greatest risk for development of asthma. REFERENCES 1. Townley RG: Mechanisms and management of bronchial asthma, in Tices: Practice of medicine. Hagerstown. Md.. 1972, Harper & Row, Publishers, vol. 1, chap. 40. 2. Townley RG, Ryo UY, Kolotkin BM, Kang B: Bronchial sensitivity to methacholine in current and former asthmatic and allergic rhinitis patients and control subjects. J ALLERG) CLIN IMMUNOL 56:429, 1975. 3. Townley RG, McGeady S, Bewtra A: The effect of Beta adrenergic blockade bronchial sensitivity to acetyl-beta-metha-
574
4.
5. 6.
7.
Townley
et al.
J. ALLERGY
choline in normal and allergic rhinitis subjects. J ALLERGY c1.1~ IS1MUNOI. 57:358, 1976. Reed CE, Townley RG: Asthma: Classification and pathogenesis. i/r Middleton, E. Reed CE, Ellis EF. editors: AllergyPrinciples and practice. St. Louis, 1978, The C. V. Mosby Company, pp. 659-677. Chai H, Farr RS, Froehlich LA, et al: Standardization of inhalation challenge. J AL.I.ERCY CLIX IMMUNOL 56:323, 1975. Townley RG, Dennis M, Itkin IH: Comparative action of acetyl-beta-methycholine, histamine. and pollen antigens in subjccta with hay fever and patients with bronchial asthma. J AI I I RGY 36:121. 1965. Townley RG. Guirgis HA, Villacorte GV, et al: Methacholine dose response curve\ in atopic and nonatopic individuals. J AI.I.L.KGY CLIN tMMUNOL
55:92,
1975.
8. Watt G. Bui T. Townley RG: Protective effect of lodoxamide tromethamine on allergen inhalation challenge. J ALLERGY (In press.) Cl-IN Ihl\lI’NOl 9. Townley RG, Nair N, Bewtra A: Comparison of inhalation responses to methacholine and histamine and their inhibition by SCH- 1000 and metaproterenol. Presented at the American Academy of Allergy, New York, 1977. IO. Curry JJ: The action of histamine on the respiratory tract in normal and asthmatic subjects. J Clin Invest 25:785, 1946. Il. Curry JJ: Comparative action of histamine on the respiratory tract tn normals, patients with hay fever, and subjects with bronchial asthma. J Clin Invest 26:430. 1947.
CLIN. IMMUNOL DECEMBER 1979
12. Curry JJ. Lowell FC: Measurement of vital capacity in asthmatic subjects receiving histamine and acetyl-hetamethylcholine: A clinical study. J ALI.ERGY 19:9. 1948. 13. Herxheimer H: Bronchial obstruction induced by allergens. histamine, and acetyl-beta-methylcholine chloride. Int Arch Allergy Appl Immunol 2:27, 195 I 14. Kang B. Townley RG, Lee CK, et al: Bronchial reactivity to histamine before and after sodium cromoglycate in bronchial asthma. Br Med J 1:867. 1976. 15. Hajos MK: Clinical studies on the role of serotonin in bronchial asthma. Acta Allergol 17:358. 1962. 16. Tiffeneau R: Cholinergic and histamine hypersensitivity of lung of the asthmatic. Acta Allergol Stsuppl.):l37, 1950 17. Spector SL, Farr RS: A comparison of methacholine and histamine inhalations in asthmatics. J AI-I ~RGI CI.I~ I\r~r~uo~. 56:308. 197.5. 18. Yu DYC. Galant SP. Gold W: Inhibition of antigen and induced bronchoconstriction by atropine in asthmatic patients. J Appl Physiol 32:823, 1972. 19. Storms WW. doPico GA, Reed CE: Aerosol SCH 1000: An anticholinergic bronchodilator. Am Rev Respir Dis 111:419, 1975. 20. Parker CD, Bilbo RE, Reed CE: Methacholine aerosol as test for bronchial asthma. Arch Intern Med 115:452, 1965. 21. Tiffeneau R: Evaluation du degre de l’asthme par une epreuve pharmacodynamique; la mesure de I’excitabilite acetylcholinique du poumon. Ann Med 56:5X2. 1955.
inhalation
challenge
studies
M.D., A. K. Bewtra, M.D., N. M. Nair, M.D., M.D., G. D. Watt, B.A., and K. M. Burke, B.S.
Omclhu, Neh.
Methacholine sensitivity has become a valuable and widely used technique for studying the irritability of the airways. Asthmatics are lOO- to l,OOO-fold more sensitive than normal subjects to various mediators such as methacholine (P-acetyl methacholine).‘-” This degree of sensitivity has been used to define asthma and also as a genetic marker.l The methacholine responsiveness may be determined by 1 of 2 methods: (1) by determining dose-response curves to increasing concentrations of methacholine while keeping the number of breaths and the volume of methacholine inhaled constant” and (2) by determining dose-response curves by keeping the concentration constant while increasing the number of inhalations of methacholine.” The first method is currently being used more widely and has been recommended by the American Academy of Allergy to provide a standard and uniform method. It is described in detail elsewhere.” The second method has been used since 1962 and has been the basis for a number of short-term and long-term studies. We have recently compared both of these methods to determine the short-term reliability of each method. The dose-response curves and thus the degree of bronchial sensitivity were determined in 19 subjects in a randomized 4-way crossover study. Each subject was challenged twice by each method at 1-wk intervals. The short-term reproducibility for both methods was good (r = 0.934 and 0.942). The correlation between methods was also significant (r =0.953). The various pulmonary function parameters that can be evaluated during an inhalation challenge are numerous and include the FVC, FEV,, SG,,,, FEF,,-,,, PEFR, flow volume loops, etc. The easiest and the most widely used currently is the FEV,, which is the minimum requirement for comparison of responses as recommended by the Standardization Committee, j From the Allergic Diseases Center, Department of Medicine, Creighton University School of Medicine. Supported in part by Grants 5P50 AI12029 and l-HR-2-2985 from the National Institutes of Health. Reprint requests to: R. G. Townley, M.D., Creighton University School of Medicine, Omaha, NE 68179. 0091-6749/79/130569+06$00.60/O
@I 1979 The C. V. Mosby
Special abbreviations used FVC: forced vital capacity FEV,: forced expiratory volume at I set SG,,.: specific airway conductance FEF25-73: PEFR:
forced expiratory flow at 25% to 75% peak expiratory flow rate
A drop in FEV, of 20% or greater from the baseline with any of the standard concentrations of methacholine is considered a positive test. As seen in Table I, we have subcategorized the degree of responsiveness with respect to dose of methacholine (inhalations) and pulmonary function (percent baseline FEV,). During the course of these studies, we found a subgroup of allergic rhinitis patients who had a decrease of 20% or greater in FEV, but who plateaued out later with increasing doses of methacholine,‘. L i as shown in Fig. 1. Some of these patient? actually showed an improvement in their pulmonary function tests with higher doses of methacholine. According to our criteria, they would have been classified as having high- or medium-positive reactions. To avoid this false high-positive classification, we found in our studies that the area under the dose-response curve is more reliable in defining the degree of sensitivity.“,
i, x
Area is calculated by integrating the best-fit parabola of the methacholine dose-response curve (Fig. 2). The test cutoff point is either a 35% drop in FEV, or 160 inhalations of methacholine. Area values that correspond to the previously defined response categories are shown in Table I. Analysis of our data in over 1,500 methacholine challenges shows the following facts: 1. Sex of the individual has no effect on the methacholine responsiveness. 2. Age does affect the methacholine responsiveness. Children and older individuals are more sensitive, and at least for research purposes, all values should be age corrected. 3. In a large group of patients, correlation of methacholine sensitivity with the histamine sensitivity is statistically significant; however, in a given individual Co.
Vol. 64, No. 6, Part 2, pp. 569-574
570
Townley
et al.
J ALLERGY
CLIN. IMMUNOL DECEMBER 1979
DEGREES OF METHACHOLINE RESPONSE
01 0
, 1020
40
METHACHOLINE
60 INHALATIONS
FIG. 1. Rapidly decreasing dose-response curves for 2 shows no change during the course of the challenge. somewhat shallower curve, which eventually exceeds strates the plateau phenomenon. His initial response 20%; however, additional inhalations did not cause a TABLE I. Characterization
Category
High Medium Low Negative
of methacholine
% Drop in FEV,
2 20% 220% 220% -=200/c
inhalation
Total no. of breaths 0.1-10
II-40 41-160 160
1 160
(5mg/ml)
asthmatics (46-Z 39-11). A normal (47-71 Subject 47-5 is an asthmatic who has a a 35% drop in FEV,. Subject 5-4 demonat 20 and 40 inhalations is greater than further drop in pulmonary function.
responses Ratio =
% Drop in FEV, No. of breaths z2.00
0.50-1.99 0.13-0.49 SO.125
Area under the curve O-306.25
306.25-1,225 1,225-4,000 4,000-5,600
Legend: We consider persons with areas of less than 306 to be high-positive responders. Areas from 306 to 1,225 are classed as medium-positive; 1,225 to 4,000 as low-positive; and 4,000 to 5,600 as negative. These classes were obtained from traditional singlepoint estimates of response, i.e., High = fall in FEVl greater than 20% after 10 cumulative inhalations of methacholine aerosol at 5 mgiml; Medium = greater than 20% after 40 cumulative inhalations; Low = greater than 20% after 160 cumulative inhalations; and Negative = less than 20% fall after 160 cumulative inhalations.
this may not be true.l. 6, 9 A person who is highly sensitive to one mediator may be less sensitive to the other and vice versa. 4. We have never seen a reaction severe enough to require hospitalizing a patient. Most patients reversed to normal pulmonary functions within 5 min following bronchodilators like isoproterenol or within 30 to 45 rnin without any bronchodilator. 5. We saw no incidences of delayed reactions to methacholine. 6. The test is reproducible and reliable over both short-term and long-term follow-up studies. To evaluate the familial nature of asthma and the methacholine responsiveness, we studied 47 atopic and 26 nonatopic families. Atopic families had at
patient,
least 1 child with current asthma. There were 648 members in these families. In 26 nonatopic families 237 members had no reported history of asthma, hay fever, or atopic eczema for at least 3 generations of first- and second-degree relatives. The proband, his or her siblings, parents, and grandparents made up the study group. All probands were excluded for genetic analysis. From these studies of methacholine responsiveness (Fig. 3), we have shown that: 1. Over 90% of asthmatics have high- or medium-positive responsiveness to methacholine. Only 3 individuals’ responses were negative, and they are former asthmatics who have been completely free of symptoms for several years. Most asthmatics who
VOLUME NUMBER
Methacholine
64
inhalation
challenge
studies
571
6, PART 2
METHACHOLINE~5mghl)
CHALLENGE
70 5 L? 50 (3
0
ALLERGIC
q q
NORMAL(ATOPIC
RHlNlTls FAMILIES)
NORMAL(NON-ATOPIC
FAMILIES)
O” 30 $ 8
a
10
aw HIGH
MEDIUM
CUMULATIVE
DOSE
as defined
NEGATIVE
RESPONSE
METHACHOLlNE
%““1 *,,: $y+bx+c;rh:ra&: Pysqr16aFIG. 3. Response IO 40 4 160 ,I 4 I
LOW
in Table I; see text for details.
(INHALATIONS)
FIG. 2. A least-squares fitted, parabolic dose-response curve. Provocative dose at 35% (PDz5) is the right-hand limit (x,,) of the integral. The X axis has been logarithmically transformed for ease of presentation.
cease to have attacks remain methacholine positive for several years after their last attack, though the degree of their sensitivity is only ‘/IO that of current asthmatics.l. 9. 1 2. Less than 5% of individuals with hay fever or nonatopic normal subjects (normals) show a highpositive response. 3. Twenty-seven percent of hay fever patients had a negative response compared to 49% of normals. 4. Hay fever patients and normals had about the same incidence of low-positive responses. 5. Thirty percent of hay fever patients had a medium-positive response compared to 18% of normals from families with a history of asthma and 8% of normals from control families. 6. The normals from atopic families had significantly fewer negative and more positive responses than normals from normal families. Chi-square analysis of our data showed that: 1. Normals from asthma families were significantly different from normals from control families (p < 0.01). 2. Hay fever patients were not significantly different from normals from atopic families (p > 0.05). 3. Asthmatics were different from all other groups. 4. Hay fever patients were different from normals of normal families only. When the frequency of positive responses for the methacholine challenge in the rzormuls from atopic and nonatopic families was determined, 2 peaks or subpopulations were obtained in the atopic (asthmatic) families (Fig. 4). In contrast, the normals from nonatopic families
0 -200
1080
Methacholine
3640
2360
5600
Area
FIG. 4. This is a frequency plot of age-corrected area under the dose-response curves for methacholine in normals from both asthma and control families. The lefthand mode shows the excess number of positive responders from asthma families.
have a single uniform distribution with no corresponding subpopulation of the methacholine responders. The individuals with a peak in the positive test may be potential asthmatics, and this bimodal distribution in the atopic families suggests a single biochemical or physical defect in asthma. The accuracy and usefulness (i.e., sensitivity and specificity) of the methacholine response test have also been evaluated (Fig. 5). Sensitivity is defined as the ratio of the true positives to the sum of the true positives plus false negatives for the whole population. Specificity is defined as the ratio of the true negatives and the sum of the true negatives plus false positives in the nonatopic population. As can be seen, as the area under the dose-response curve cutoff value
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FIG. 5. Comparing response to methacholine with clinical 1,300 optimized both sensitivity and specificity at 90%.
‘“lC’I,&CH?L,NF
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H,STAMhE
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FIG. 6. A graph of the mean (+ SEM) provocative doses of methacholine and histamine for 20% and 35% falls (PD2,, and PD,,) following placebo, SCH 1000, and metaproterenol.
increases, the sensitivity continues to increase and specificity continues to decrease. The best sensitivity and specificity were obtained at an area of 1,300; however, the range 800 to 2,000 was acceptable. Since the low-positive responses range from 1,225 to 4,000, one has to be very careful in interpreting results beyond 2,000 for fear of falsely labeling them positive. Figs. 6 and 7 show the results of the effects of 40 pg 8-isopropylnoratropine methabromide (SCH 1000) or atrovent or 1.3 mg metaproterenol or placebo on methacholine and histamine challenges in 10 subjects with mild-to-moderate asthma. These challenges were performed in a double-blind and randomized fashion. This study was done using method
‘b
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CLIN. IMMUNOL DECEMBER 1979
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No. 1, i.e., increasing concentrations of methacholine and histamine administered via a dosimeter.” The results in Fig. 6 show that: 1. Methacholine at 1.31 + 1.0 and 8.6 + 7.4 mg was required to produce 20% and 35% decreases in FEV, , respectively. In the presence of SCH 1000, 30.3 t 7.9 (p < 0.01) and 47.9 t 6.5 mg (p < 0.001) and, in the presence of metaproterenol, 17.6 +- 6.3 (p < 0.02) and 32.3 2 8.9 (p < 0.05) mg methacholine produced the same responses, respectively. 2. Histamine at 3.8 -+ 2.2 and 13.7 ? 6.8 mg was required to produce 20% and 35% drops in FEV,. In the presence of SCH 1000, 5.9 t 2.9 and 16.0 -+ 7.2 mg (p is not significant) and, in the presence of metaproterenol, 15.5 ? 4.2 (p < 0.02) and 33.9 & 8.0 (p = 0.05) mg histamine produced the same responses, respectively. 3. SCH 1000, an anticholinergic compound, antagonizes methacholine responses specifically while metaproterenol, a /3-adrenergic agonist, antagonizes both the histamine and methacholine bronchoconstriction. These studies indicate that the histamine bronchoconstriction in asthmatics is not due to a vagal reflex, as SCH 1000 should then have blocked the effects of both methacholine and histamine. DISCUSSION From our experience of performing over 1,500 methacholine challenges in patients with bronchial asthma since 1962 and in an additional 500 nonasthmatic persons, either atopic or nonatopic, we think methacholine sensitivity should be part of the definition of asthma. We have not observed a single nega-
VOLUME NUMBER
64 6, PART 2
tive inhalation challenge to methacholine in a patient who currently has bonafide bronchial asthma. ‘. A. ’ Among current asthmatics, the severity of asthma determines the bronchial sensitivity of subjects to methacholine. This sensitivity varies from 100 to several thousand times that of normal subjects .2. 6*“I- ” However, in former asthmatics, the degree of bronchoconstriction to methacholine was also related to the severity of past asthma symptoms.‘. “L I2 The mean sensitivity of former asthmatics is approximately ‘/IO that of current asthmatics. In a number of studies. the airway response to inhaled histamine has been found to be similar to that after methacholine.g, 12. I:‘. I.‘. I6 Those patients who are extremely sensitive to methacholine are also sensitive to histamine, although Spector and Farr” do report that a few patients sensitive to methacholine are only slightly sensitive to histamine and vice versa. Histamine is much more effective in producing bronchoconstriction when administered by aerosol than by the intravenous route, even though the intravenous route produces many more systemic responses such as flushing and throbbing headaches.” Many stimuli appear to cause reflex bronchoconstriction in asthmatic subjects in concentrations that evoke little or no response in healthy subjects. In man, Yu et al.‘# reported that atropine by intravenous or aerosol route markedly inhibits the bronchial constrictor response to inhaled antigen in asthmatic patients. Atropine and some recent atropine derivatives such as SCH 1000 (atrovent) are potent bronchodilators in many patients with asthma.lg Our results (Fig. 7) show SCH 1000 to result in some bronchodilation, but this was somewhat less than that produced by metaproterenol. Although SCH 1000 has been found to markedly inhibit the bronchoconstriction induced by methacholine, it has little or no effect in inhibiting histamine-induced bronchoconstriction. Thus our findings do not support the concept that histamine sensitivity is due to a heightened vagal reflex. In contrast, metaproterenol showed comparable protect’on against both methacholine and histamine. This suggests that these mediators are able to cause bronchoconstriction because they are not opposed by the intact P-adrenergic receptors that normally serve to maintain bronchodilatation. Subjects with allergic rhinitis and atopic eczema, as well as other members of asthmatic families, are more likely IO develop asthma.‘, i, “‘3 2’ However, which of these individuals will eventually develop asthma is not known. Those subjects with allergic rhinitis and atopic eczema or who are siblings of asthmatics and who have a high or medium response to methacholine
Methacholine
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may be the ones at greater risk. By periodically studying these high-risk subjects before and after the pollen seasons and after respiratory infections, it may be possible to relate their methacholine sensitivity to the onset of asthma. Our results show that 30% of allergic rhinitis patients who were family members of an asthma family and 18% of nonatopic members of these same families had a medium-positive response to methacholine. This group, plus the 5% of hay fever subjects with a high-positive methacholine response represent the population who may be at greatest risk for development of asthma. REFERENCES 1. Townley RG: Mechanisms and management of bronchial asthma, in Tices: Practice of medicine. Hagerstown. Md.. 1972, Harper & Row, Publishers, vol. 1, chap. 40. 2. Townley RG, Ryo UY, Kolotkin BM, Kang B: Bronchial sensitivity to methacholine in current and former asthmatic and allergic rhinitis patients and control subjects. J ALLERG) CLIN IMMUNOL 56:429, 1975. 3. Townley RG, McGeady S, Bewtra A: The effect of Beta adrenergic blockade bronchial sensitivity to acetyl-beta-metha-
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4.
5. 6.
7.
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et al.
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choline in normal and allergic rhinitis subjects. J ALLERGY c1.1~ IS1MUNOI. 57:358, 1976. Reed CE, Townley RG: Asthma: Classification and pathogenesis. i/r Middleton, E. Reed CE, Ellis EF. editors: AllergyPrinciples and practice. St. Louis, 1978, The C. V. Mosby Company, pp. 659-677. Chai H, Farr RS, Froehlich LA, et al: Standardization of inhalation challenge. J AL.I.ERCY CLIX IMMUNOL 56:323, 1975. Townley RG, Dennis M, Itkin IH: Comparative action of acetyl-beta-methycholine, histamine. and pollen antigens in subjccta with hay fever and patients with bronchial asthma. J AI I I RGY 36:121. 1965. Townley RG. Guirgis HA, Villacorte GV, et al: Methacholine dose response curve\ in atopic and nonatopic individuals. J AI.I.L.KGY CLIN tMMUNOL
55:92,
1975.
8. Watt G. Bui T. Townley RG: Protective effect of lodoxamide tromethamine on allergen inhalation challenge. J ALLERGY (In press.) Cl-IN Ihl\lI’NOl 9. Townley RG, Nair N, Bewtra A: Comparison of inhalation responses to methacholine and histamine and their inhibition by SCH- 1000 and metaproterenol. Presented at the American Academy of Allergy, New York, 1977. IO. Curry JJ: The action of histamine on the respiratory tract in normal and asthmatic subjects. J Clin Invest 25:785, 1946. Il. Curry JJ: Comparative action of histamine on the respiratory tract tn normals, patients with hay fever, and subjects with bronchial asthma. J Clin Invest 26:430. 1947.
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12. Curry JJ. Lowell FC: Measurement of vital capacity in asthmatic subjects receiving histamine and acetyl-hetamethylcholine: A clinical study. J ALI.ERGY 19:9. 1948. 13. Herxheimer H: Bronchial obstruction induced by allergens. histamine, and acetyl-beta-methylcholine chloride. Int Arch Allergy Appl Immunol 2:27, 195 I 14. Kang B. Townley RG, Lee CK, et al: Bronchial reactivity to histamine before and after sodium cromoglycate in bronchial asthma. Br Med J 1:867. 1976. 15. Hajos MK: Clinical studies on the role of serotonin in bronchial asthma. Acta Allergol 17:358. 1962. 16. Tiffeneau R: Cholinergic and histamine hypersensitivity of lung of the asthmatic. Acta Allergol Stsuppl.):l37, 1950 17. Spector SL, Farr RS: A comparison of methacholine and histamine inhalations in asthmatics. J AI-I ~RGI CI.I~ I\r~r~uo~. 56:308. 197.5. 18. Yu DYC. Galant SP. Gold W: Inhibition of antigen and induced bronchoconstriction by atropine in asthmatic patients. J Appl Physiol 32:823, 1972. 19. Storms WW. doPico GA, Reed CE: Aerosol SCH 1000: An anticholinergic bronchodilator. Am Rev Respir Dis 111:419, 1975. 20. Parker CD, Bilbo RE, Reed CE: Methacholine aerosol as test for bronchial asthma. Arch Intern Med 115:452, 1965. 21. Tiffeneau R: Evaluation du degre de l’asthme par une epreuve pharmacodynamique; la mesure de I’excitabilite acetylcholinique du poumon. Ann Med 56:5X2. 1955.
