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* From the Department of Pediatrics, Wake Forest University School of Medicine, Winston-Salem, NC
Correspondence to: Bruce K. Rubin, MD, FCCP, Professor and Vice Chair for Research, Department of Pediatrics, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157-1081; e-mail: brubin{at}wfubmc.edu
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Abstract |
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 TOP Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Design: Sputum or simulants were analyzed untreated and after the addition of the test agent at 1:5 volume to volume ratio for a contact period of 60 s. The concentrations of the agents were as follows: guaifenesin, 20 mg/mL; iodinated glycerol, 3 mg/mL; surfactant (Exosurf; Glaxo Wellcome; Research Triangle Park, NC) containing 13.5 mg of phospholipid per milliliter; albuterol, 5 mg/mL; and amphibian Ringer's solution (ARS) as a control. Dynamic viscoelasticity and surface mechanical impedance were measured in a magnetic microrheometer. Cohesiveness was measured using a filancemeter. The wettability of a hydrophilic surface was measured using an image processing system. The mucociliary transportability of sputum was timed on the frog palate, and cough transportability (CTR) was measured in a cough machine.
Results: When compared to sputum that had no test agent or ARS added, all agents reduced sputum elasticity G', with surfactant, albuterol, and guaifenesin significant at p < 0.001. As well, guaifenesin (p = 0.006), albuterol (p = 0.003), and surfactant (p = 0.02) decreased surface mechanical impedance (frictional adhesiveness) compared to untreated sputum. However, there were no significant changes in wettability, hydration, cohesiveness, or CTR with any agent, and there were no significant changes in the properties of sputum or simulants treated with test agents when compared to those treated with ARS. Guaifenesin irreversibly disrupted mucociliary transport when applied directly to the frog palate.
Conclusions: These agents appear to have a minimal direct action on sputum in vitro, suggesting that at the concentrations studied, these agents do not have a significant beneficial effect on either the mucociliary transportability or CTR of chronic bronchitis sputum. However, there could be an effect of some of these agents after oral administration, especially if there is a secondary effect of the agent on an effector cell.
Key Words: chronic bronchitis • mucolytics • sputum • surfactant
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mechanisms of action of mucoactive agents are often unclear, and they could relate to the effects on the secretory and mucociliary apparatus or to direct effects on the secretions.2 3 4 The purpose of this study was to determine the in vitro effects of several agents on the properties of sputum collected from 30 adults with stable chronic bronchitis. We deliberately did not evaluate classic, direct-acting mucolytic agents such as thiol derivatives (N-acetyl-L-cysteine) or dornase alfa (Pulmozyme; Genentech; San Francisco, CA), because we wished to determine if nonmucolytic but putatively mucoactive agents would have a direct effect on sputum or mucus simulants. We hypothesized that we might detect the direct effects of these agents on the expectorated bronchitis sputum, especially as these relate to mucociliary and cough clearability.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1. Guaifenesin is a secretagogue that is readily available in many over-the-counter cough and cold medications. The concentration of guaifenesin used was 20 mg/mL, corresponding to the maximum airway concentration after oral dosing.5 This agent is thought to enter the airway secretions unmetabolized and to have a direct effect either on the mucus or the epithelium.2 5
2. Iodinated glycerol (IPG) has been reported to be a mucolytic in vitro, but was shown to have no effect on mucus properties or pulmonary function in vivo.6 The concentration of IPG used was 3 mg/mL, corresponding to the maximum calculated airway concentration after IV dosing.7
3. The surfactant used (Exosurf; Glaxo Wellcome; Research Triangle Park, NC) has been demonstrated to alter the active surface properties of respiratory mucus and to enhance mucociliary clearance.8 The concentration of phospholipid (DPPC) in the surfactant was 13.5 mg/mL, corresponding to the reconstituted concentration for aerosol administration in the neonate.
4. Albuterol nebulizer solution is a ß2-agonist that has been shown to enhance ciliary beat frequency and power, and possibly to promote mucociliary clearance.9 Albuterol is also thought to induce mucin secretion in the large airway.10 The concentration of albuterol used was 5 mg/mL, corresponding to the concentration of albuterol in the nebulizer solution.
5. In order to preserve mucociliary clearance on the mucus-depleted frog palate, all medications were prepared in amphibian Ringer's solution (ARS) with an osmolarity of 206.5 mOsm/L containing the following: NaCl, 98.3 mmol/L; KCl, 2.7 mmol/L; and CaCl2, 1.5 mmol/L. This solution served as a control.
Mucus Simulant Studies
Mucus simulants were prepared by crosslinking locust bean gum
(2% and 4% weight/volume), dissolved in ARS using 0.02 mol/L
sodium tetraborate
(Na2B4O7).
To stabilize the mucus simulant and to raise the solids composition to
levels seen in chronic bronchitis sputum, sucrose at 4% weight/volume
was added to the solution before crosslinking. Three 2-mL
aliquots of these two simulants with well-characterized physical and
transport characteristics were placed in a 5-mL, wide-mouthed container
and layered with 0.5 mL of the test solution for a contact period of
60 s at 37°C. The supernatant was removed by careful pipetting,
and the mucus simulant was then tested for viscoelasticity,
cohesiveness, hydration, surface mechanical impedance, wettability,
mucociliary clearability, and cough clearability. Each experiment was
repeated with three test samples for a total of 30 analyses. The
testing was performed at an ambient temperature (24 ± 2°C).
Studies of Expectorated Sputum
Expectorated sputum was collected from 30 subjects > 16 years
old with chronic bronchitis as defined by the American Thoracic Society
criteria.11
The patients participating in this study were
in stable condition and had required no adjustment in their medications
in the previous 6 months. None of the patients were taking oral
corticosteroids, mucoactive medications, oral or inhaled
anticholinergic medications, theophylline, or ß-agonist medications.
Two patients were taking nasal corticosteroids. None of the patients
were taking inhaled corticosteroids or oral antihistamines. Other
medications used included antihypertensive medication in five patients,
cholesterol-reducing agents in seven patients, and insulin in one
patient. Patients with tuberculosis, HIV infection, or active
hemoptysis were excluded.
Sputum was collected by direct expectoration into a sterile cup over a 30-min period during the course of pulmonary function testing. The patients were asked to swallow all saliva before expectorating, and dental cotton was placed between their lips and gum as previously described by Puchelle and colleagues.12 The sputum was then visually separated from any remaining saliva before being divided into five aliquots of 200 µL each. This was accomplished using a Teflon-tipped, positive-displacement pipette that allows accurate collection of a specific volume of mucus without altering sputum properties.3 4 The aliquots were placed in airtight containers fitted with an O-ring and stored at -70°C until analyzed. Because mucin and DNA polymers may be susceptible to degradation with freezing and thawing, it is the viscoelastic properties of sputum that would be most sensitive to this handling. However, freezing and thawing in this manner have been demonstrated to have minimal effects on sputum viscosity or elasticity.13 14 The sputum was analyzed untreated and after the addition of the test agent at 1:5 volume to volume ratio for a contact period of 60 s at 37°C. The testing was performed at an ambient temperature (24 ± 2°C).
The Physical Properties of Secretions
Viscoelasticity (Rheology): In the magnetic
microrheometer, a dissecting microscope was used to position a
small steel ball, 100 µm in diameter, in a 4-µL sample of mucus;
this was then placed in the field of an electromagnet, where it was
oscillated at driving frequencies of 1 and 100 rad/s. The image of the
ball was magnified and projected onto photocells, where the magnitude
of displacement of the ball and its phase lag with respect to the
driving force are used to calculate the dynamic loss modulus
(viscosity) (G'') and the storage modulus (elasticity) (G') of the
specimen. Mechanical impedance "rigidity" (G*) is the
vectorial sum of viscosity and elasticity, and the loss tangent
"recoil" (tangent 
) is the ratio of G'' to G'. Both are
calculated from the viscoelastic data.4
Cohesiveness: Cohesiveness (spinnability) is the thread-forming ability of mucus under the influence of large-amplitude elastic deformation. This was measured in millimeters using the filancemeter. The measurement was performed with a 25-µL mucus sample at a distraction velocity of 10 mm/s. An electric signal conducted through the mucus sample was interrupted at the point where the stretched mucus thread was broken. This distance represents the mucus cohesiveness.15
Sputum Hydration (Percent Solids): The sputum samples were weighed in a microbalance and then dried by lyophilization overnight. The dried sample was weighed again to calculate the percent solids composition.
The Transport Properties of Secretions
In Vitro Cough Transportability: A simulated cough
machine was used to measure the airflow-dependent clearability of
sputum. A model Plexiglas trachea, rectangular in cross-section
(1.2 x 2 cm) was connected to a 6.4-L tank containing air
pressurized to 12 psi, giving a flow rate of about 11 L/s. A solenoid
valve controlled the air release through a flow-constrictive element
that mimicked the airflow pattern of a natural cough. A sinusoidal
constriction (length, 7.7 cm; height, 8 mm) was used to decrease the
airway diameter while minimizing the turbulence of the system. A sample
(volume, 40 µL; depth, 0.5 mm) was placed in a thin line across the
base of the Plexiglas trachea. The bulk transport of the sample was
measured in millimeters after a single cough maneuver. Three successive
measurements were made, and the results were averaged.16
In Vitro Mucociliary Transportability: Using hypothermia as anesthesia, a mature northern leopard frog was rapidly decapitated, the jaw was disarticulated, and the palate was removed by cutting through from the junction of the posterior pharynx and esophagus out to the skin of the back. The excised palate was placed on a piece of gauze saturated with ARS. The palate was placed in a dish loosely covered with plastic wrap and allowed to rest in a refrigerator at 4°C for 12 to 18 h to deplete the mucus.
The following morning, the palate was placed in a box with a fitted-glass top. Humidity was maintained at 95 to 100%, and the temperature was kept at 22 to 24°C. The palate was focused under a microscope so that a 12.7-mm micrometer scale ran between the optic bulges to the opening of the esophagus. The movement of a 4-µL sputum specimen was timed as the trailing edge moved across a 7.62-mm segment. Three measurements of mucus transport rate were taken to minimize variability, and the average transport rate was normalized to the transport rate for collected endogenous frog mucus.17
The Surface Properties of Secretions
Sputum Surface Mechanical Impedance by the Rolling Ball
Technique: The rolling ball technique is used in industry to
measure the adhesiveness of glue strips. The magnetic microrheometer
and computer measurement software were modified so that we could
make rolling ball adhesion measurements on small mucus specimens. The
steel microprobe was placed on the surface of the mucus layer without
using a paraffin oil cover. The magnetic force needed to roll the
sphere across the surface of the sample was used to calculate the
surface G* at 1 rad/s. This is a measurement of frictional
adhesion.18
Sputum Wettability (Contact Angle 
): A 20-µL drop of
test material was gently placed onto the surface of a glass microscope
slide cleaned with chromic sulfuric acid and rinsed with deionized
water. It was then stored in absolute ethanol to maintain dehydration
before use. A stabilization time of 1 min was allowed before capturing
the image of the drop in the video processing
system.19
20
Data Analysis
Statistical analysis of the data was performed using a
statistics package (StatView 5; SAS Institute; Cary, NC). The
relationships between the physical and transport properties of mucus or
sputum and how these relate to in vitro pharmacologic
treatment were examined using the equality of variance F test.
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The in vitro mucociliary transportability on the frog palate (MCTR) of mucus simulants was severely decreased after guaifenesin to less than one third of the untreated or control-treated values. It was subsequently shown that guaifenesin irreversibly disrupted MCTR when applied directly to the frog palate.
Studies of Expectorated Sputum
As shown in Table 1
, all agents reduced G', compared to untreated secretions, with
surfactant, albuterol, and guaifenesin significant at p < 0.001.
This result was associated with a decrease in viscosity and, for
guaifenesin and IPG, a decrease in hydration (increased percent
solids). However, these changes were not significant (NS) when compared
to ARS treatment. Surfactant, albuterol, and guaifenesin all decreased
G* when compared to untreated sputum (p < 0.001), but again
these changes were NS when compared to the ARS treatment. There were no
significant changes in wettability, cohesiveness, or cough
transportability (CTR) with any agent when compared to ARS treatment
in vitro.
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mucus is a viscoelastic gel consisting primarily of water and high-molecular-weight, crosslinked glycoproteins that form a tangled network. Respiratory mucus is usually cleared by cilia. Sputum, which is mucus mixed with inflammatory cells, cellular debris, and often bacteria, is generally cleared by coughing. The rheology of mucus is its capacity to undergo flow and deformation.4 A true solid responds to a stress with a finite elastic deformation that is totally recovered after the stress is removed. A liquid responds to a stress with viscous deformation, flowing continuously for the time that the stress is applied. After the removal of the stress, the flow ceases and there is no recovery of the strain. A viscoelastic gel such as mucus initially stores energy; with continued stress, it will begin to flow like a liquid. The viscoelastic behavior of mucus was thought to be one of the major determinants of mucociliary clearance. Studies using mucus simulant gels (made from crosslinked vegetable gums) suggest that MCTR on the frog palate is impeded by increasing mucus G*, but increases with greater mucus recoil.21 However, the relationship between the properties of actual airway secretions and MCTR is unclear. Giordano and colleagues22 studied the tracheal mucus velocity of dogs and found a negative correlation between the tracheal clearance rate and the elasticity of mucus secreted in a tracheal pouch. Recent studies using human airway secretions have shown little relationship between MCTR and rheology,23 and almost no relationship with in vitro CTR in the cough machine.24
There is increasing recognition that the surface interaction of secretions with the epithelium is most critical in regulating mucus transport, especially by coughing.8 16 18 20 This is consistent with the fluid dynamics of low mechanical impedance, non-Newtonian liquids (like mucous gels), where airflow-dependent transport is most influenced by interfacial interactions and is far less dependent on stress-strain (viscoelastic) characteristics.
Surfactant phospholipids may increase mucociliary transport and ciliary beat frequency. Allegra et al25 evaluated mucociliary transport on the frog palate after either saline solution or surfactant obtained from pig lung was sprayed on the excised palate. Saline solution induced a constant decrease in transport rate (p < 0.01), while the surfactant caused an increase of approximately 16% (p = NS). The difference between the two treatments was highly significant (p < 0.001). This could be due to the surfactant increasing the efficiency of energy transfer from the beating cilia to the mucus layer by reducing mucociliary frictional energy loss. In vitro experiments have shown that the addition of surfactants to cystic fibrosis sputum reduces tenacity and increases clearability.26 The high adhesion tension and abnormal surface properties of sputum suggest that an aerosolized surfactant could be an effective mucokinetic therapy for patients with inflammatory airway disease. We tested this hypothesis in a multicenter, placebo-controlled trial of aerosolized surfactant in 66 patients with stable chronic bronchitis who were randomized to receive surfactant and 21 patients who were randomized to receive saline solution treatment. The patient demographics among groups were similar at the baseline. In patients who received 607.5 mg of surfactant for 2 weeks, prebronchodilator FEV1 increased from 1.22 ± 0.08 L at baseline to 1.33 ± 0.09 L at day 21 (p < 0.05); postbronchodilator FEV1 improved 10.4% by days 14 and 21 (p < 0.05); and the ratio of residual volume to total lung capacity, a measure of thoracic gas trapping, decreased 6.22% by day 21 (p < 0.05). In the surfactant group, there was a dose-dependent increase in sputum MCTR.8
The effect of the agents tested in this study did not appear to be mediated by direct action on sputum or sputum simulants alone, and, as anticipated, none had specific mucolytic properties. At the concentrations studied, these agents do not seem to have a physiologically significant direct beneficial effect on either the mucociliary or cough clearability of chronic bronchitis sputum.
Mucolytics are assumed to act directly on secretions. Although we showed an in vitro reduction of elasticity with some of these agents, there was no change in viscosity in contradistinction to data reported earlier by Braga and colleagues,27 who found a reduction in viscosity but not elasticity, albeit with S-carboxymethylcysteine, an agent not tested in these studies. Other researchers28 have reported no change in viscoelasticity or clinical function with the use of mucolytic therapy. Based on in vitro studies, Puchelle et al29 suggested that when using mucoactive agents, for optimal sputum clearance, the specific ratio of viscosity to elasticity is important. The viscoelasticity of many of the sputum samples studied was in this range, but unlike the Puchelle et al29 study, we did not find an "optimum" for transport. In fact, there was little relationship between viscoelasticity and transport, as we have previously reported.4 24 In this study, for MCTR, R2 was < 0.02, and for CTR, R2 was < 0.01 for both G' and G'' by linear regression analysis (data not shown).
In our studies of sputum transportability, only in vitro exposure to IPG increased MCTR, suggesting a direct effect on the ciliated epithelium. This is consistent with the direct effects of potassium iodide on the ciliated epithelium.30 Although albuterol has been consistently shown to increase ciliary beat frequency, there is no clear evidence that this is reflected in an increase in mucus transport, with some investigators9 finding improved in vivo mucociliary transport after ß-agonist administration, while other groups31 32 found no such increase. The fact that we did not see any increase in MCTR in vitro strongly suggests that any in vivo improvement on mucociliary clearance after albuterol administration is not mediated by a direct effect on the cilia, but may instead be secondary to increased mucociliary activity induced by mucus secretion.10
However, the topical administration of guaifenesin, even mixed with sputum, seems to paralyze cilia irreversibly. This may well be due to the oily and hypertonic nature of the guaifenesin that we tested, as indicated by the significant increase in the solids content of the secretions after exposure. However, because guaifenesin is meant to be orally administered and not topically applied or inhaled, the effects of guaifenesin therapy on sputum clearance could be different when the medication is administered as directed. Because both guaifenesin and IPG are thought to be secreted unchanged into the airway surface liquid, and sputum concentrations of these agents have been directly measured, we presumed that in vitro exposure of mucus to these agents would mimic airway exposure in vivo. It is likely that this is an oversimplification, and thus the clinical efficacy or lack of efficacy of these medications after oral dosing cannot be extrapolated from the data presented herein.
In conclusion, it is known that classic mucolytic agents can reduce the viscosity of abnormally viscous secretions. It is useful to evaluate this action both in vitro and in vivo to prevent the over thinning of secretions with a concomitant reduction in mucus clearance.33 Possibly some nonmucolytic, mucoactive agents can alter the transportability properties of sputum by acting directly on the secretions. However, these data strongly suggest that in vitro assessments cannot be a substitute at this time for clinical trials in evaluating the effectiveness of these agents.
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Acknowledgements |
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Footnotes |
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For related article see page 201.
Manuscript received September 15, 1998; revision accepted January 15, 1999.
Abbreviations: ARS = amphibian Ringer's solution; CTR = cough transportability; G' = storage modulus (elasticity); G'' = loss modulus (viscosity); G* = mechanical impedance "rigidity"; IPG = iodinated glycerol; MCTR = in vitro mucociliary transportability on the frog palate; NS = not significant
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This article has been cited by other articles:
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  P. V. Dicpinigaitis and Y. E. Gayle Effect of Guaifenesin on Cough Reflex Sensitivity Chest, December 1, 2003; 124(6): 2178 - 2181. [Abstract] [Full Text] [PDF]
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