Dilutions should be made with 0.9% sodium chloride for injection, using empty, sterile, type I borosilicate glass vials, shaking after adding the diluent until a clear solution is obtained. This task should preferably be performed by the hospital Pharmacy Department. Methacholine dilutions in isotonic saline solution greater than 0.125mg/ml are stable for 3 months when stored at 4°C.28,31 If the solvent contains a preservative (e.g. 0.4% phenol to inhibit bacterial growth), the stability is reduced and the solution should be used within two weeks. The pH of the methacholine in the saline solution is moderately or weakly acidic, depending on the concentration of the methacholine itself.28,31 Reconstituted vials should be stored in cool conditions (4°C), avoiding exposure to sunlight, and used at room temperature. No special precautions are required for disposal of excess product.30Table 2 shows the schemes for preparing dilutions.

This requires the use of a Wright jet impactor nebuliser or similar.36 Once the baseline spirometry has been performed, the patient should be asked to breathe quietly through a mouthpiece connected to the nebuliser while seated comfortably, with a nose clip in place. Alternatively, the use of a mask may be considered, in this case also using a nose clip. The test begins by inhaling the diluent and continues with the administration of increasing methacholine concentrations (from 0.03mg/ml to 16mg/ml) (Table 3). At each stage of the test, 3ml of the relevant concentration are placed in the nebuliser, and inhaled continuously by the patient while tidal breathing for 2min. After each inhalation, the patient should be removed from the equipment and after 30–90s, a spirometry should be performed (not more than three manoeuvres during the following 3min). If the FEV1 falls by 20% or more, the BPT is concluded. Otherwise, administer the following concentration until this decrease is reached or the remaining preparations have been used.27,28 To assure the cumulative effect of the doses, there should be no more than 5min between the administration of one concentration and the next. For safety reasons, once the test is finished, a short-acting ß2 adrenergic agonist must always be administered (salbutamol or terbutaline), preferably with a pressurised cartridge, in order to return the patient to his or her baseline situation.

The diluent may be used to familiarise the subject with the technique and to identify the existence of bronchial lability. A greater than 10% fall in the FEV1 after inhaling the diluent indicates a positive response.28 The post-diluent FEV1 will be the reference value for the comparison following the administration of the different methacholine concentrations.

Since the whole procedure is quite time-consuming, modifications have been proposed that enable the BPT to be commenced with doses of bronchoconstriction agent that are higher than those recommended in the original protocol. Thus, the first concentration to be administered is decided considering the presence of respiratory symptoms, usual medication, baseline FEV1 and its fall after administering the diluent (Table 4).36 If there is no significant fall in the FEV1 (decrease less than 5% with respect to the reference value) after the first methacholine concentration and there are no respiratory symptoms, a dose may be skipped, omitting the concentration immediately afterwards.37 This simplified protocol should only be carried out by experienced technicians.

The main limitation of this method lies in its dependence on the respiratory pattern. In fact, variations in both the respiratory rate and tidal volume amplitude can modify the dose administered.

This method uses a jet nebuliser connected to a dosimeter, programmed to nebulise for the first 0.6s of each inhalation, delivering an output of 9µl±10% in that period.27,28 In this method, the diluent and 5 increasing methacholine doses (from 0.0625 to 16mg/ml) are administered (Table 3).38

At end exhalation during tidal breathing, at functional residual capacity (FRC), the subject should be connected to the mouthpiece and must inhale slowly (for 5s) until total lung capacity (TLC). Once maximum inspiration has been reached, the apnoea should be maintained for 5s before exhaling slowly. This procedure should be repeated with intermediate periods of quiet breathing until 5 inhalations have been completed for each concentration, within a period of not more than 2min. There should be no longer than 5min between the administration of each concentration level so that a cumulative effect can be produced.27,28 The spirometry should be performed in the 30–90s following the inhalation of each concentration. The sequence of steps to be followed until the test is completed is identical to that previously described.

It has been suggested that the deep inhalations and apnoea phases that characterise this procedure may have a bronchoprotective effect in normal subjects and patients with mild hyperresponsiveness.39 Therefore, although there are no major differences between these two methods in most asthma patients, in subjects with mild hyperresponsiveness, deep inhalations may produce false negatives when using the dosimeter method with respect to the tidal breathing method.40,41

These minimise the number of concentrations to use, thereby reducing the technician's work and possible error sources.

The Chinn dosimetric method was designed for the European Community Respiratory Health Survey II, as an abbreviated protocol (Table 3).42 It was developed for a Mefar dosimeter, which programmed for a nebulisation time of 1s, has an output of 0.01ml of aerosol each time it is activated.43 Each inhalation sequence requires that, once connected to the mouthpiece in FRC, the patient inhales slowly until TLC, maintaining apnoea for 3s, before disconnecting the mouthpiece immediately afterwards. The slow inspiration and subsequent apnoea should total at least 6s if possible. Once the baseline spirometry has been performed, at least three inhalations of the diluent should be taken, repeating the spirometry at 60s. If the FEV1 falls by >10% after the diluent, then the long protocol should be followed (Table 3). In the simplified protocol, 4 concentrations should be used to administer 5 doses, performing the spirometry 60s after each dose level, and interrupting the test if a =20% fall in the FEV1 with respect to the post-diluent value is recorded.42,43 A bronchodilator should be administered after the last dose in this case as well.

The Schulze dosimetric method is an even greater simplification, as it uses a single concentration (16mg/min) to generate 5 increasing doses (Table 3) using APS dosimeters, which allow both pulse and continuous nebulisation.44–46 While there are no differences between both procedures in adults, continuous nebulisation is recommended in children due to the greater irregularity of the respiratory cycle.46 In this method, the nebulisation is performed while the patient is tidal breathing, with a direct view of his or her respiratory tracing. To guarantee better aerosol deposition in the airways, the inspiratory flow should not exceed 0.5l/s.46 Two minutes after the administration of each dose, the spirometry is carried out, maintaining the same performance and end of procedure characteristics as in the other protocols.

Adenosine is an endogenous nucleoside involved in numerous physiological processes (control of vascular and intestinal tone, neurotransmission, neurosecretion, etc.) and inflammatory reactions.54 Synthesis takes place inside the cells from the hydrolysis of adenosine 5'-monophosphate (AMP) by 5'-nucleotidase, or by the catabolism of S-adenosyl homocysteine. When the intracellular AMP concentration increases (hypoxia, cell activation), it crosses to the outside for reuptake by the cell itself, or to be bound to membrane cytoplasmic receptors (A receptors).54–57 High adenosine concentrations have been detected in the bronchoalveolar lavage and exhaled breath condensate of patients with asthma and chronic bronchitis, compared with healthy individuals, and experimental models show that adenosine is released into the blood as a result of bronchial provocation with allergens.55,56

The bronchoconstrictive action of inhaled AMP is a result of the release of histamine, interleukin 8, leukotriene C4 and other mediators from the mast cells, platelets, neutrophils and epithelial cells, once their A2B receptors have been activated.54,55 Other mechanisms such as neurosensory mechanisms, the axon reflex and cholinergic stimulus also appear to be important.54,55,58

For the BPT, due to its higher solubility in saline solution, the disodium salt of AMP (adenosine 5'-monophosphate sodium salt, Sigma Chem., St. Louis, MO, USA) is used, which is dispensed as a dry crystalline powder. The purity grade of the product is 99%–100%.2 The powder preparation is stored in the refrigerator with a dry desiccant. Adenosine is a class C medicinal product for pregnancy.

Addresses of interest: Adenosine distributor: Sigma–Aldrich Química, S.A., Calle Ronda de Poniente 3, 2. Tres Cantos 28760, Madrid. Contact telephone number: 900101376. Fax: 900102028. Website: http://www.sigmaaldrich.com/spain/acerca.html. Email: esorders@sial.com.

This substance is a hexahydrate sugar alcohol (C6H14O6), with a molecular mass of 182.17g/mol, and is a stereoisomer of sorbitol.63,64 Up to the 1920s, it source was manna, a product derived from heating the bark of the Fraxinusornus tree. It is currently produced by high pressure hydrogenation, but manufacture using fermentation processes is already underway.63,65 It possesses osmotic properties and has been used for decades as a food additive (in diet foods and chewing gum). It was also introduced some years ago in the medical field to relieve intracranial hypertension, replace blood plasma and as a diuretic.63 When inhaled, mannitol dehydrates the bronchial mucosa, which is accompanied by an increase in osmolarity in the mucosa and release of inflammatory mediators from the mast cells, with a profile very similar to that evoked by adensosine.17,64,66,67 Its use as a bronchial provocation agent was approved in 2006 in Australia and later in the European Union (2007), Asia and the United States (2010). It was authorised in Spain in July 2008.

In the Spanish market, for the analysis of BH, mannitol is marketed in the form of dry powder capsules with preset doses (Osmohale™).

Data on the use of Osmohale™ during pregnancy are limited. Animal studies do not suggest direct or indirect harmful effects in terms of reproductive toxicity. However, the effects of a possible hyperreactivity reaction on the mother and/or foetus are unknown, so Osmohale™ should not be administered to pregnant women.68 With respect to lactation, no effects are expected in breastfeeding infants, since systemic exposure to mannitol in breastfeeding mothers is insignificant. Studies with oral mannitol do not suggest effects on fertility either.68

Addresses of interest: Osmohale™ distributor: Aldo-Unión, S.A. Baronesa de Maldá 73. Esplugues de Llobregat 08950, Barcelona. Contact telephone number: +34933727111. Fax: +34933716198. Website: http://www.aldo-union.com/. Email: info@aldo-union.com. General test information: http://www.aridol.info/spain.

The mannitol test requires less equipment and is much more simple than the other indirect tests (particularly those related with exercise), providing reasonably superimposable results.63 The Osmohale™ kit contains 19 capsules (one without active substance, three capsules containing 5, 10 and 20mg, respectively, and 15 capsules containing 40mg) together with a dry powder inhaler (Inhalator™, Boehringer Ingelheim, Germany). The FEV1 is generally used to record the response.68

The steps to follow are as follows68: (a) with the patient seated comfortably, apply a nose clip and instruct them to breathe through the mouth; (b) insert the 0mg capsule into the device. Puncture the capsule by depressing the buttons on the sides of the device carefully once only, since a second puncture may shatter it; (c) the patient should exhale completely away from the inhaler and then should bend their head slightly forwards, holding the device at 45°, to perform the inspiration manoeuvre which should be deep, rapid and short; (d) at the end of the inspiration, start a 60s timer; the patient should hold his or her breath for 5s and exhale through the mouth before removing the nose clip; (e) at the end of the 60s, perform spirometry, measuring the FEV1 at least in duplicate to obtain two reproducible measurements; the highest reading will be the baseline FEV1 value; (f) insert the 5mg capsule in the inhaler and proceed as above; and (g) the following doses to be administered will be: 10mg, 20mg, 40mg, 80mg (two consecutive 40mg capsules), 160mg (four 40mg capsules) and 160mg (four 40mg capsules); the test should be stopped when a total dose of 635mg is reached or the patient has a positive response.

After inhalation of each dose, the capsule should be checked to ensure that it is empty. A second inhalation should be performed if it has not been completely emptied.68 After completion of the test, a standard dose of a fast-acting ß2-adrenergic agonist should be administered. The test duration is variable and depends, logically, on whether BH is detected (17±7min) or not (26±6min).69

A response is considered to be positive when68: (a) there is a =15% fall in FEV1 with respect to the baseline value (using the FEV1 value after the administration of 0mg as a comparator) (PD15); or (b) there is a =10% incremental fall in FEV1 between two consecutive doses. Some studies recommend using the fall threshold of 10% (PD10) calculated by interpolation of the log-linear dose–response curve and thereby classifying BH in asthmatic subjects into severe (0–75mg), moderate (75–315mg) or mild (315–635mg).70

The repeatability of the mannitol test is similar or better than other BPTs that use drugs, both in adults67,71 and in children.72,73 However, after repeated tests within a short space of time, there is a refractory period (the same occurs with exercise-induced bronchoconstriction) due to a phenomenon of tachyphylaxis in the response of the ASM to contracting mediators released from the mast cells.74

The mannitol test translates into the intensity of asthmatic airway inflammation to a large extent, is modified by the action of inhaled corticosteroids and its PD15 maintains a close relationship with: (a) the responses evoked by other indirect stimuli (adenosine, exercise, eucapnic hyperpnoea and hypertonic saline); and (b) the amount of eosinophils in sputum and the FENO.63,66 The specificity of mannitol in the diagnosis of asthma is above 95%, its sensitivity is around 60% and its predictive values depend on the pretest probability of asthma.63,66,75 The sensitivity increases to almost 90% when patients on steroid treatment are excluded.69

Thus, the mannitol BPT is presented as a very interesting tool when evaluating the effectiveness of anti-asthmatic treatment and establishing the degree of control reached, and even for monitoring the evolution of occupational asthma.18,63,66,76 At the same time, the technique facilitates the collection of bronchial secretions (which increase after the test), providing a further source of analysis of airway inflammation at a given time, if desired.77

Finally, but no less importantly, the inhaled mannitol test is an alternative to consider for the diagnosis of exercise-induced asthma with an efficacy similar to that obtained with eucapnic hyperventilation.63,78,79

In this respect, the International Olympic Committee has approved its use for the study of BH in elite athletes, and to justify the use of ß2-adrenergic agonists before a sporting event.64

Mannitol is a safe compound; the most commonly observed side effects are cough (83%), which may sometimes cause interruption of the test, and headache.68,69 The tusigenic effect is associated with asthma, regardless of the bronchoconstriction, and tends to decrease after inhaled steroid treatment. Headache usually appears more often in subjects with a negative test, probably due to the high number of forced expiration manoeuvres performed.68,69 The complete list of its adverse effects is shown in Table 8.

General precautions relative to performing spirometries and BPTs as listed in extenso in previous sections should be observed for the mannitol test, including extra precautions in patients with haemoptysis of unknown origin, pneumothorax and recent abdominal or thoracic surgery.68

Physical exercise provokes bronchospasm in a significant proportion of asthmatic patients.80 It is also the only trigger in a group of patients who play sports or have an occupation that requires a high level of physical effort, thus defining the entity known as exercise-induced asthma (EIA). The prevalence of EIA has been estimated at 40%–90% in asthmatics, 10%–50% in elite athletes, 20%–35% in ice skaters, 30%–50% in Nordic ski competitors, 15% in long distance runners and 11%–28% in competitive swimmers.81 Particularly relevant predisposing factors are atopy (especially in long distance runners), a high concentration of pollutants from the combustion of fossil fuels in the inhaled air and the inhalation of chloramines in swimming pools. If the presence of a reversible obstruction has not been documented, EIA must be diagnosed using an exercise bronchial provocation test (EBPT) or other properly accredited alternative bronchial provocation test.28,81,82

The development of exercise-induced bronchospasm (EIB) has been justified by three pathogenic hypotheses: the heat, osmolar and capillary theories. The heat theory attributes bronchospasm to an adrenergic reflex triggered by temperature loss in the airways.83 The hyperventilation caused by exercise also increases the periciliary osmolarity, due to dehydration of the airway surface liquid, which could favour the degranulation of mast cells and release of inflammatory mediators.84 Finally, the hyperaemic or capillary theory proposes that microvascular vasoconstriction due to cooling of the airway wall during exercise is followed by rapid reheating in the recovery period, causing reactive hyperaemia and oedema of the airways.85 Since none of these physiopathological theories fully explains the phenomenon, it has been suggested that the pathogenic mechanism may be different for each individual, type of exercise and environmental condition.

In half of patients, EIB has a refractory period consisting of a decrease in the bronchoconstrictive response when the exercise is repeated within an interval of under 2h.80 This phenomenon, which may be crossed between various indirect stimuli,16,71 was attributed to the depletion of inflammatory mediator deposits by the mastocytes.16 However, controversy remains over its origin, and more recent hypotheses consider that it may be due to the absence of a response to the hyperosmolar stimulus in repeated exercise.86

The aforementioned mechanisms that trigger bronchoconstriction in response to intense exercise can be simulated with eucapnic hyperventilation.99

Inhalation of non-isotonic saline aerosols can cause bronchoconstriction due to the release of endogenous mediators that cause contraction of the ASM and oedema.17 Changes in the airway osmolarity caused by these types of aerosols promote the release of mediators, such as histamine, leukotrienes and prostaglandins,114,115 as well as causing neural alteration, with release of neuropeptides and an increase in the parasympathetic tone.116 In fact, drugs that reduce the release of mast cell mediators or that modify non-cholinergic neural activity, such as nedocromil or furosemide, play a protective role against hypertonic saline BH,27 as occurs with inhaled corticosteroids.117

For the hypertonic saline BPT, it is recommended to use 4.5% saline solution, which should be administered using ultrasonic nebulisers, as they generate a more dense aerosol than jet nebulisers.27 It also requires a two-way non-rebreathing valve and a connection tube with the nebuliser, which must have smooth inner walls and constant length and diameter. The valve should have a saliva collector, since the test usually causes profuse salivation. The ultrasonic nebuliser used should generate an aerosol flow of at least 1.2ml/min and have an easily disassembled cup with a capacity for 100–200ml of solution. The most commonly used nebulisers (Mistogen and De Vilbis Ultraneb) provide a flow of 1.5–3ml/min. In any case, it should be determined by weighing the cup and tubing before and after a nebulisation. Once the nebuliser flow has been established, the dose administered can be determined in an operating period.

The increase in the hypertonic saline dose is obtained by gradually increasing the nebulisation times. In the first step, the subject is nebulised for 30s, then 1min, 2, 4 and 8min in the following steps. The bronchoconstrictive response is measured at 30–90min using spirometry, and if the fall in FEV1 exceeds 10% with respect to the baseline value, the last exposure time is repeated instead of proceeding to the next step.27 The test is considered to be positive when there is a >15% fall in the FEV1,118,119 at which point it should be discontinued. A bronchodilator should be administered both in this case and if the test is ended after reaching the maximum dose.

The bronchial response to hypertonic saline solution is expressed using a dose–response curve, which shows the change in the FEV1 (percentage fall with respect to the baseline value) against the cumulative dose of aerosol administered (in ml). The PD15 is determined by linear interpolation, enabling the severity of the BH to be classified into mild (PD15>6ml), moderate (PD15: 2.1–6.0ml) and severe (PD15<2.0ml).120 There is still insufficient information to recommend the systematic determination of bronchial reactivity indices.117,121

The hypertonic saline BPT may be useful for the diagnosis of asthma, reaching a sensitivity of 85% in adults and 75% in children, with a specificity of almost 100%.122,123 The response to hypertonic saline has a notable relationship with the presence of respiratory symptoms and the use of rescue medication.117,124 Moreover, in the detection of exercise-induced bronchospasm, it may reach results similar to exercise provocation,125 and even slightly higher in children.7 Nevertheless, in many cases, its clinical use may be compromised with the introduction of the mannitol BPT.