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asthmatic and nonasthmatic subjects, we compared changes in methacholine and isoproterenol respon siveness before PAF inhalation and for six days follow.
Effect of PAF-acether inhalation on nonspecific bronchial reactivity and adrenergic response in normal and asthmatic subjects. R J Hopp, A K Bewtra, M Nabe, D K Agrawal and R G Townley Chest 1990;98;936-941 DOI 10.1378/chest.98.4.936 The online version of this article, along with updated information and services can be found online on the World Wide Web at: http://chestjournal.chestpubs.org/content/98/4/936

Chest is the official journal of the American College of Chest Physicians. It has been published monthly since 1935. Copyright1990by the American College of Chest Physicians, 3300 Dundee Road, Northbrook, IL 60062. All rights reserved. No part of this article or PDF may be reproduced or distributed without the prior written permission of the copyright holder. (http://chestjournal.chestpubs.org/site/misc/reprints.xhtml) ISSN:0012-3692

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Effectof PAF-AcetherInhalationon NonspecificBronchialReactivityand AdrenergicResponsein Normaland AsthmaticSubjects* Russell I. Hopp, D. 0; Againdra K. Bewtra, M. D.; Makoto Nabe, M.D.; Devendra K. Agrawal, Ph.D.; and Robert G. Townley, M.D. , F.C.C.P

Bronchial hyperreactivity, although recognized as a hail

reactivity nor isoproterenol responsiveness was changed

mark

following

of asthma,

is not

totally

understood.

Mast

cell

derived mediators, including histamine, have been shown to cause immediate bronchoconstriction, but until recently, no single mediator has been shown to induce prolonged changes in airway reactivity. Recent reports indicate PAF acether (PAF) can induce increased nonspecific bronchial reactivity in normal subjects but not in asthmatics. We sought to elucidate the role ofPAF in airway hyperreactivity

by comparing the effect of inhaled PAY on methacholine and isoproterenol airway responsiveness in six nonasthmatic and six asthmatic subjects. Neither nonspecific airway

P

AF-acether important

(PAF) has been reported role

in

the

pathogenesis

to have an of asthma,'

predominantly because PAF has been shown to in crease nonspecific bronchial reactivity in humans.2'3 Cuss et al2 reported that nonasthmatics had increased airway reactivity after PAF inhalation. Rubin et al@did not find that asthmatics had increased airway respon siveness after PAF inhalation. The period of observa tion after PAF inhalation was different in these two studies. To further clarify the effect of inhaled PAF in asthmatic and nonasthmatic subjects, we compared changes in methacholine and isoproterenol respon siveness before PAF inhalation and for six days follow ing PAF inhalation. SUBJECTS

AND METHODS

Subjects Twelve subjects were studied, including six nonasthmatics and six asthmatics, ages 22 to 44 years. Demographic data are presented in Table 1. Asthmatics discontinued antihistamine therapy two days

PAF

inhalation

in the

nonasthmatic

subjects

in

the six days following PAF. Asthmatics had increased airway responsiveness to methacholine at two hours post-PAF, which

did not persist.

Responsiveness

to isoproterenol

did

not change in the asthmatic subjects. Additional evaluation of the role of PAF in causing changes in airway reactivity is warranted.

(Chest

1990; 98:93641)

PAFPAF-acether; pFEF6O-S0forced partial expiratory at 60 to 80 percent remaining from the baseline VC

flow

inhaled five breaths of 200 mgfL of PAF (30 @gdelivered at the mouth) at 8 AM. A summary of the protocol is included in Table 2. On each study day, a bill vital capacity maneuver was performed prior to methacholine and isoproterenol, followed by a 15-minute wait. Three before saline solution and after saline solution partial

Table 1—DemographkData CategoryAge, (Ib)Normal24

yr

155Normal31 175Normal32 138Normal40 154Normal21 165Normal44 110Mean32±8.8 149±23Asthma25 160Asthma31 118Asthma22 170Asthma28 175Asthma21 170Asthma23 150Mean25±3.8 157±21Table

Sex

Ht (cm)

M M M M M F

70 70 65 66 72 60 67±4.4 74 60 73 72 72 69 70±5.2

M F M M M M

Wt

prior to and during the two-week protocol, but they could take inhaled @-agonistsas needed, except for 8 hours prior to study days. The protocol was approved by the Institutional Review Board of Creighton University, Omaha, Neb, and all subjects signed a consent form.

Protocol A 15-day protocol was performed on each ofthe 12 subjects. The protocol was identical at all days except on day 9 when the subject *From the Allergic Disease Center, Creighton University School of Medicine, Omaha. Manuscript received February 5; revision accepted March 26. Reprint requests: Dr Hopp, Allergic Disease Center, Creighton

University, Omaha 68178 @

936

ProtocolDayStudy 2—PAYinhalation PMMonday1 2Tuesday2 2Wednesday3 2Monday8 2Tuesday9 2Wednesday10 2Thursday11 2Monday15

Day

PAF

Methacholine, AM

—¿ —¿ —¿ —¿ +(8 AM) —¿ —¿ —¿

10 10 10 10 10 10 10 10

Isoproterenol,

2

Effect PAF-AcetherInhalationin Asthma(HoppetaI@I

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FIGURE

13,JTrn

1. Representation

ofthe

measurement

and

calculation

forced vital capacity maneuvers were then performed, followed by the respective challenge. At each methacholine or isoproterenol step, only partial vital capacity maneuvers were performed (see “¿ChallengeMethods―). After the methacholine and isoproterenol challenge, a repeat full vital capacity was performed. Challenge Materials and Methods

CI6PAF(Bachem Inc. Torrance,CA)was dissolved in 100 percent ethanol and diluted in phenol-buffered saline solution and 0.25 percent human serum albumin. The final concentration of ethanol in the administered C,6PAF was less than 2 percent. Methacholine chloride (J.T Balcer Chemical, Phillipsburg, NJ)challenge was done at doses of2.0, 20, and 60 mglml, with ten breaths at the first two doses and five breaths (x 2) at 60 mglml for a cumulative total of 24.60 mg (820 breath units) for the normal subjects. The methacho line challenge for the asthmatics was done at doses ofO.2, 0.6, 2.0,

-:@@@:1[ii@EP1S

of pFEF6O-80.

and 20 mg/mI, with ten breaths per dose. The cumulative total of methacholine for asthmatics was 6.84 mg (228 breath units). One breath of 1 mg/ml of methacholine equals 1 breath unit. The inhalation of the saline solution diluent preceded the inhalation of methacholine. A timed inhalation method@ (from a DeVilbiss 646 nebulizer, the DeVilbiss Company, Somerset, PA) was employed. In brief, the subjects slowly inhaled medication starting from 25 percent of the inspiration capacity to 75 percent of inspiratory capacity, which ensures a consistent inhalation pattern for all doses.' The output of the nebulizer averaged 0.03 ml per actuation. Isoproterenol inhalation was performed using a five-step regimen. Doses included the following: 0.052, 0.157, 0.471, 1.41, and 4.24 mg/mI. Saline solution diluent preceded the isoproterenol chal lenge. The timed inhalation method was also used for the isopro terenol challenge.@

Table 3—PulmonaryFUnCtIOn Results pre-PAF° AsthmaticsNormalsDay1238pj1238p13.62±0.653.59±0.66

pre-M FEy, Fbst-M FEY1 pt Pro-Iso FEY, Fbst-Iso FEY, pt

tSignificance tSignificance

3.66±0.653.50±0.64NS3.37±0.853.61±0.943.48±1.013.28±0.77NS 2.54±0.78

2.51±0.46

2.58±0.53

2.34±0.36

NS

3.02±0.73

3.37± 1.0

3.03±0.79

2.72±0.69

NS

0.015 3.84±0.28

0.003 3.86±0.41

0.000 3.48±0.56

0.002 3.38±0.56

NS

0.008 3.48±1.06

0.018 3.62±1.00

0.051 3.44±0.90

0.004 3.44±0.84

NS

3.98±0.81

4.15±0.11

3.89±0.76

3.75±0.70

3.60±1.16

3.69±1.01

3.41±0.91

3.52±0.85

NS

0.015

0.149

0.018

0.058

0.1

0.1

0.8

0.008

Iso = isoproterenol. by paired t test between before and after challenge by ANOVA among days for changes in FEV,.

FEy,.

CHEST I 98 I 4 I OCTOBER, 1990

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937

@

Baseline pulmonary function was measured using a respirometer (Collins 13.5-L Respirometer, Warren E. Collins Inc. Braintree, Mass). The forced vital capacity (FYC) and forced vital capacity at is (FEY,) were calculated. Determination of the changes in pul monary function caused by methacholine or Iso were calculated from a forced partial expiration curve. This was performed at 50 to 60 percent of the baseline vital capacity (VC). Full VC maneuvers were not performed during the challenge with methacholine or isoproterenol to avoid the bronchodilating effect of such maneuvers. The forced partial expiratory flow rate at 60 to 80 percent remaining from the baseline VC (pFEF6O-80) was calculated from the partial volume/time curve (Fig 1). After saline solution, and at each challenge step, triplicate pFEF6O-80 measurements were performed at each dose until the end of the respective challenges. The mean of the three pFEF6O80 values at each step was used to determine changes in pFEF6O80. The partial flow-volume measurement was done three minutes after each dose of the challenge agent. The results of the methacholine challenges are expressed as the area under the dose-response curve to a maximum of a 35 percent fall in pFEF6O-80. This is determined using a trapezoid method

0

2.00

0

(0

0@

1.00

Baseline

I

I

T

5

10

15

MINUTES FIGURE

inhalation

2.

Change

on study

in

pFEF6O-80

immediately

day 9. Asthmatics = open

following

circles;

PAF

normals

= closedcircles

(area =[x+y/2xz].'Theareawasnecessary, asnormal subjects

often did not have a 35 percent change in pFEF6O-80 to methacho line. The dose of methacholine was expressed in log units. The results of the isoproterenol challenge are expressed as the total area under the dose-response curve without a top response restriction, using the same trapezoid method.' The dose of isoproterenol was expressed in log values. A measurement ofpFEF6O-80 was determined before and 5, 10, and 15 minutes after PAF on day 9. In addition, total and differential white blood cell counts were determined before and 5, 10, and 15 minutes after PAF inhalation. The total white blood cell count was determined using an automated counter (Baker 8000, Serono Baker Diagnostics, Allentown, PA). The differential blood counts were obtained by reading 200 cells. An analysis ofvariance (ANOVA) was used to compare the change in methacholine response and isoproterenol response, baseline FEy, values, and baseline pFEF6O-80 before and after PAF on the different days. Paired t tests were used to compare results between comparable study days (eg, study day 1 and study day 9). A p value of0.05 percent or less was considered as significant. The change in bronchial response to methacholine and isoprote renol was also compared by determining the expected lowest methacholine and isoproterenol area and expected lowest percent age change from baseline to isoproterenol and methacholine.6 This expected value was then compared, using a paired t test analysis, with the lowest actual value post-PAF inhalation.

methacholine or isoproterenol. Asthmatics, as ex pected, had significant FEY, responses to methacho line (228 breath units of methacholine) and had significant improvements after isoproterenol on two of four days. Normal subjects also had responses, as measured by FEy,, to methacholine (820 breath units of methacholine), but not to the same extent as asthmatics; and they had a significant response to isoproterenol on one of four days. There was no significant difference in day-to-day variability in base line FEy, in either group, as determined by ANOVA. Figure 2 presents the change in pFEF6O-80 after PAF inhalation (day 9). Measurements were taken pre PAF and 5, 10, and 15 minutes post-PAF. There was

not a significant fall in pFEF6O-80 following PAF inhalation in either group. The results for the blood count measurements are presented in Table 4. There was a significant fall in total white blood cell count and absolute neutrophils, at 5 minutes, when corn pared with baseline. There was a rebound in neutro phil numbers and total white blood cell count above baseline at 15 minutes, which was signfficantly differ ent than baseline. The fall in the eosinophil count was not significant and the lymphocytes did not change. Table 5 contains the FEY, results before and after methacholine and isoproterenol on the four days post PAF inhalation in normals and asthmatics. Using an ANOVA, there was no significant difference among the post-PAF days for baseline FEY, values, post methacholine FEY, values, pre-isoproterenol FEY,

RESULTS

The results are divided into asthmatic and normal, pre-PAF and post-PAF components. Table 3 shows the mean FEy, values for the asthmatics and normal subjects during the pre-PAF arm, before and after

Table 4—Effectofinhaled PAYon Circulating White Blood Cell Count* mmTotal5,386±

Baseline5

mm10

mm15

1,178t5,880±2,0677,093±2,897tNeutrophils3,016.7 1,2694,040± 2,846tLymphocytes1,736+4521,788±4281,864±6291,825±392Eosinophils304.8±250202.8±162225±184252±196Basophils29±4545±4124±3447±52 1 ±1,6514,744.4± ±1,2001,837.6 ±1,177t3,568.

6Mean ±SD, cellslm' tp