Plasma Histamine and Catecholamines in Stable ... - Clinical Science

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Dec 8, 1981 - Departments of Medicine and Clinical Pharmacology, Royal Postgraduate Medical School, Hammersmith Hospital, London. (Received 19 June ...
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Clinical Science (1982) 62,661-665

Plasma histamine and catecholamines in stable asthmatic subjects P . J. B A R N E S , P . W. I N D A N D M. J . B R O W N Departments of Medicine and Clinical Pharmacology, Royal Postgraduate Medical School, Hammersmith Hospital, London (Received 19 June 1981; accepted 8 December 1981)

Summary 1. Venous plasma histamine and catecholamines were measured in stable asthmatic subjects by a recently developed specific and sensitive radioenzymatic assay. 2. Plasma histamine concentrations were significantly elevated in both extrinsic and intrinsic asthmatic subjects, compared with both normal controls and patients with chronic obstructive airways disease. There was no correlation between histamine concentration and severity of airways obstruction, however. 3. Elevated plasma histamine concentrations at rest may indicate increased release of mediators from ‘leaky’ mast cells in asthma. 4. Plasma catecholamine concentrations in asthmatic subjects did not differ from normal and there was no correlation with severity of bronchoconstriction or with plasma histamine concentration.

Key words: asthma, catecholamines, histamine, mast cell mediators. Introduction

Histamine is released from sensitized human lung 11, 21 and from basophils [31 on antigen challenge in vitro. Histamine causes bronchoconstriction by a direct action on bronchial smooth muscle and indirectly by vagal reflex mechanisms. Whether histamine released from pulmonary mast cells can be detected in the circulation is less certain, however. Fluorimetric assays for histamine, which have been available Correspondence: Dr Peter Barnes, School of Medicine, Cardiovascular Research Institute, University of California, San Francisco, CA 94143, U.S.A. 0143-522 1/82/060661-05S01.50/1

for many years [41, are too insensitive to measure the low concentrations of histamine in human plasma. More recently radioenzymatic assays with greatly improved sensitivity have been used to measure histamine in plasma; with such assays increased concentrations of plasma histamine have been reported in asthmatic subjects during antigen challenge [SI and during spontaneous attacks [6,71, although resting values were not significantly different from normal. Using a radioenzymatic assay for plasma histamine with increased sensitivity and precision, we have examined the resting concentrations of plasma histamine in asthmatic, chronic bronchitic and non-asthmatic control subjects. Mast cells contain other bronchoconstrictor mediators, including prostaglandin D,, leucotrienes and bradykinin, which may all contribute to bronchospasm in asthma [81. Since there is no evidence that these mediators may be differentially released it is possible to use plasma histamine as a marker of mediator release in uivo. Adrenergic agonists are the most effective and widely used bronchodilators in the treatment of asthma. Conversely b-adrenoceptor antagonists cause increased bronchoconstriction in asthmatic [9-111 but not in normal subjects [12]. This suggests that there may be increased padrenergic stimulation of asthmatic airways. Since there is no convincing evidence for direct sympathetic innervation of human airways [131, it is likely that circulating endogenous catecholamines, particularly adrenaline, play an important role in the regulation of airway tone in asthma. As with histamine, it is not possible to measure plasma catecholamines, particularly adrenaline by fluorimetric assay, but the introduction of sensitive radio-enzymatic assays [14, 151 has allowed the measurement of endogenous catecholamines in

@ 1982 The Biochemical Society and the Medical Research Society

P.J . Barnes, P . W. Ind and M . J. Brown

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human plasma. We have used a modification of these assays with improved specificity and sensitivity to study resting adrenaline and noradrenaline concentrations in clinically stable asthmatic subjects. Catecholamines may act directly on airway /.-receptors to produce bronchodilatation, or may act on mast cell /.-receptors to reduce the release of bronchoconstrictor mediators [16, 171. We have therefore also examined the relationship between adrenaline and histamine concentrations in plasma. Methods

Subjects We studied patients with asthma demonstrated by a > 20% increase in forced expiratory volume in 1 s (FEV,) after inhaled bronchodilator, and sputum or blood eosinophilia (0.3 x lo9 cells/l). They were classified into ‘extrinsic’ [multiple positive immediate reactions to skin prick tests with a battery of common allergens, and elevated serum immunoglobulin E (IgE) concentrations1 or ‘intrinsic’, with negative skin tests and normal serum IgE concentrations. All asthmatic subjects were in a stable clinical state with no exacerbation within 4 weeks of study. We also studied patients with symptomatic chronic bronchitis and irreversible airflow obstruction who had negative skin tests. All patients were selected from the Chest Clinic, Hammersmith Hospital, and were normally ambulant before blood sampling. Normal subjects with no history of asthma were selected from amongst medical and laboratory personnel, and were classified into atopic (multiple positive skin tests) or non-atopic (Table 1). Normal subjects were matched in age to the extrinsic asthmatic group, so that an effect of age on plasma catecholamine concentrations was avoided. We have found no effect of age on plasma histamine concentrations (unpublished

observations). None of the patients was taking oral medication, and none had taken oral steroids within 2 months of sampling. All use of inhalers was stopped at least 4 h before sampling. In all subjects FEV,., was measured by dry spirometry (MacDermott, Garw Instruments, Glamorgan, U.K.) and the highest of three consecutive forced expiratory manoeuvres recorded. Blood was sampled betwen 09.00 hours and 15.00 hours in all subjects, so that the effect of circadian variations in plasma histamine and catecholamine concentrations was not significant. A sampling cannula (Butterfly 19 g, Abbott Laboratories) was inserted into a forearm vein and the subject rested supine for at least 15 min before blood was drawn. Blood was collected into pre-chilled plastic tubes containing 5 ml of ethylenediaminetetra-acetic acid (0.5 mol/l) for histamine estimation and into lithium-heparin tubes (5 ml) for catecholamine assay. Plasma was separated by centrifugation at 4OC within 30 min of collection, and stored at -8OOC until analysed.

Assays Plasma catecholamines were measured by a recently developed double-isotope technique modification of the catechol 0-methyltransferase (COMT) assay, which allows both high precision and sensitivity [181. Separate aliquots of each sample were incubated with COMT (prepared from rat liver) and either S-[3Hladenosylmethionine or S-[14Cladenosylmethionine (The Radiochemical Centre, Amersham, Bucks, U.K.). Noradrenaline and adrenaline standards (100 ng/ml) were also added to the latter aliquots and converted into I14Clmetanephrines, which were added as tracers to the 3H tubes after incubation. The final recovery of 14C corrects both for incomplete methylation and for losses during extraction and during thin-layer chromatography. The sensitivity of this assay was 0.06

TABLE1. Details of subjects before measuremen1 of plasma catecholamines and histamine Normal

No. Sex Age (years) Mean & SEM Range 4)predicted FEV, Mean t SEM Range

Asthmatic

Chronic bronchitic

Non-atopic

Atopic

Extrinsic

Intrinsic

20 All male

6 All male

26 21 male

I2 5 male

6 male

27.2 i 2 . 0 15-42

31.2 i 0 . 6 0 29-33

26.5 & 2.3 14-64

48.4 i 4 . 2 19-71

59.2 f 2.1 46-69

99.3 C 1.7 89- I I 5

98.3 C 4.1 88- I10

59.1 C 4 . 3 23-96

53.3 5 . 2 25-90

+

46.9 f 7.5 15-85

10

Histamine and catecholamines in asthma

nmol/l for both noradrenaline and adrenaline. Intra- and inter-assay coefficients of variation were 4.6% and 9.8% respectively. Plasma histamine was measured by a similar double-isotope modification of our previously described radioenzymatic method [ 191. Histamine N-methyltransferase was prepared from rat kidney. Sensitivity was 0.2 nmol/l and intra- and inter-assay coefficients of variation were 6.3% and 13.4% respectively. This assay had a sensitivity much greater than any previously reported method 1201. Results were analysed by Student's t-test for unpaired samples, and by Spearman's rank correlation test. Results In normal subjects (n = 26) plasma histamine was 2.42 f 0.17 nmol/l (mean +_ SEM) and there was no significant difference between non-atopic (n = 20) and atopic (n = 6) subjects (Fig. 1). In patients with extrinsic.asthma (n = 26) plasma histamine was significantly higher than in agematched normal controls (5.72 f 0.38 nmol/l; P < 0.01). There was no significant correlation between plasma histamine concentration and severity of asthma as measured by % predicted FEV,., (r = 0.004). Similarly, in patients with intrinsic asthma (n = 12) plasma histamine P