- Email: [email protected]
Clinical application of transepithelial potential difference measurements in cystic fibrosis
Clinical application of transepithelial potential difference measurements in cystic fibrosis Russell A. S a u d e r , MD, S a r a h E. C h e s r o w n , MD, a n d G e r a l d M. L o u g h l i n , MD From the Departments of Pediatrics, Anesthesia, and Critical Care Medicine, The Johns Hopkins Medical Institutions, Baltimore, and the Department of Pediatrics, University of Florida, Gainesville
We studied transepithelial potential difference (PD) in normal persons, patients with chronic disease, and patients with cystic fibrosis (CF), using the technique described by Knowles and co-workers. A maximal PD value (PDmax) and an a v e r a g e PD value (PDmean) were determined for e a c h study of the nasal respiratory epithelium. The v o l t a g e response to superfusion of 10 -4 mol/L amiloride onto nasal mucosa was noted. The PD of the palm, wrist, and between two fingertips was also measured. Nasal PDmax and PDmean of the CF group were more negative than the control (P <0.01) and chronic disease groups (P <0.01). After application of amiloride, the voltage c h a n g e in nasal PD was greater in the CF group than in the non-CF control groups (P <0.01). There were no clinically significant differences in the PD of the palm, wrist, or fingertips of the three groups. These d a t a confirm the observation that patients with CF have hyperpolarized nasal epithelia that demonstrate greater c h a n g e in response to amiloride than that in non-CF controls. These results indicate a possible role for the use of in vivo nasal PD measurements as a diagnostic test for cystic fibrosis. (J PEDIATR1987;111:353-8)
in vivo measurements of transepithelial potential difference, originally described in dog trachea by Boucher et al., 1 has recently been extended to humans by this same group. 2,3 Application of this technique to the study of cystic fibrosis has identified an abnormality in chloride transport across the epithelium of the respiratory tracP -6 and the sweat duct. 8-~~In addition to abnormal chloride transport, nasal respiratory epithelium in patients with CF Supported by American Lung Association of Florida, Pediatric Pulmonary Center Grant MCJ-2013, and New Investigation Research Award HL-26952-03 from the National Heart, Lung, and Blood Institute. Presented in part at the Annual Meeting of the Cystic Fibrosis Club, Anaheim, Calif., 1984. Submitted for publication Feb. 25, 1987; accepted May 5, 1987. Reprint requests: Gerald M. Loughlin, MD, The Johns Hopkins Hospital, Department of Pediatrics, CMSC 141, 600 N. Wolfe St., Baltimore, MD 21205.
demonstrates an increased rate of sodium absorption. 4-7'~l In vivo PD measurements in patients with C F have been used extensively by this group as a research tool, 8,12-14but these findings have not been confirmed fully in an independent clinical laboratory. Is The objectives of our study were (1) to confirm the differences in nasal PD between a group of C F patients and normal control patients or patients with CF PD
Cystic fibrosis Potential difference
[
a variety of chronic diseases, (2) to determine the difficulties and limitations of applying the technique of measuring in vivo nasal PD in a routine clinical setting, and (3) to explore other easily accessible epithelial surfaces for possible diagnostic differences between CF patients and non-CF controls.
353
354
Sauder, Chesrown, and Loughlin
The Journal of Pediatrics September 1987
-I00.
---e----
+2SD
........
-2SD
-80. ,
PDMAx
(mv)
-60' -40-
- ~-"~ ....
+2SD
9
........
+2SD
_J_k i ll ___e_____2SD
CT
It .........
CD
2SO
CF
Fig. 1. Maximum potential difference (PDmax, mean + 2 SD) for nasal epithelium in control group (CT), Patients with chronic disease (CD), and patients with cystic fibrosis (CF). Note there is no overlap in data for CF vs CD or CT groups.
METHODS Transepithelial potential difference measurements were obtained in a study population made up of 35 volunteers divided into three groups. A normal healthy control group consisted of 17 healthy nonsmokers (12 female) ranging in age from 15 to 39 years (average 26.7 years). A chronic disease control group consisted of five volunteers (three female) ranging in age from 7 to 38 years (average 20 years). The volunteers in the chronic disease group had a variety of chronic illnesses, including ulcerative colitis, rigid spine syndrome, mitral valve prolapse, reactive airway disease, and paroxysmal atrial fibrillation; two had chronic obstructive pulmonary disease with bronchiectasis of unknown cause. Sweat tests by pilocarpine iontophoresis gave normal results. Thirteen patients (10 male) known to have cystic fibrosis made up the CF group; the diagnosis of CF was confirmed by quantitative pilocarpine iontophoresis and clinical historyJ 6 The mean age of the CF group was 21 years (range 11 to 26 years). Clinical severity had no role in selecting CF patients for measurement of PD. One heterozygote, the mother of a patient known to have CF, was studied and included in the normal control group. All procedures were approved by the Institutional Review Board of the University of Florida College of Medicine. Informed consent was obtained from each patient or their parents before entering the study. Transepithelial PD measurements were obtained in a manner similar to that described in dogs and other animals by Boucher et al. H and in humans by Knowles et a13 3 PD was measured between a fluid-filled double-lumen, vinyl exploring catheter (inner diameter 0.71 mm, outer diameter 1.5 mm) (Dural Plastics & Eng PTY Ltd, Dural, New
South Wales, Australia) and a subcutaneous reference electrode. Previous studies have shown that the subcutaneous space is isoelectric with the submucosal space of airway epithelia. 14,17.18 The reference electrode was constructed by filling a sterile 23-gauge butterfly needle (Abbott Laboratories, Hospital Products Division, North Chicago, Ill.) with sterile lactated Ringer solution in 4% agar. The exploring catheter was continuously perfused with lactated Ringer solution (Travenol Labs, Inc., Deerfield, Ill.) at a rate of 0.2 to 0.4 mL/min. Both the subcutaneous reference electrode and the exploring catheter were connected by matched calomel electrodes (calomel reference electrodes, Fischer Scientific, Pittsburgh) to the input of a high-impedance, low-resistance buffer amplifier (Donald Martin, University of Florida). The output of the buffer amplifier was connected to a highimpedance digital multimeter (Dynasian Corp.) and a strip chart recorder (Moseley Autograf). PD measurements of the palm, the volar surface of the wrist, and between two fingertips were obtained by touching the fluid-filled exploring catheter to each site and recording the PD values as the average of two to four individual measurements. Nasal PD was measured along the inferior border of the inferior turbinate under direct vision using an otoscope. The exploring bridge catheter was advanced through the inferior meatus, and measurements were obtained at 0.5 cm intervals posterior to the anterior meatus, using the tip of the anterior turbinate as a landmark. An average nasal PD (PDmean) was determined for each nostril, and a total PDmean was determined for each study. A maximum nasal PD (PDmax) was recorded as the maximum stable PD measurement recorded either during entry or with-
Volume 111 Number 3
Nasal potential difference in cystic fibrosis
35S
-80"
-70-
PDMEAN
(my)
---ir---§
-60-
oo
-50-
__.LL
oo
-40-
-50-20"
-I0-
eeS ---~----.t.2S D
III 9 8 9
CT
........ 9
-2SD
--*2SD
9
-2SD
........
CD
-2SD
CF
Fig. 2. Mean potential difference (PDmean, _+ 2 SD) for nasal epithelium. Value for one patient from normal control group (CT) is within 2 SD of mean of cystic fibrosis group (CF). CD. Chronic disease group.
drawal of the exploring bridge catheter. PDmax was determined in each patient for the right and left nostrils, and for the total study. Values for baseline PD measurements were accepted if PD measurements were stable for >3 seconds and if the PD measurement at the anterior turbinate and the palm of the hand were comparable at the beginning and end of the study. After baseline PD values were obtained, the double-lumen exploring catheter was repositioned at the site of the PDmax value. Once the stable PDmax value was relocated, amiloride hydrochloride 10-4 mol/L dissolved in lactated Ringer solution was perfused at a rate of 4 ml/min for 4 minutes through the second lumen of the exploring catheter from a separate perfusion system. The change in PDmax that resulted was recorded and expressed as both an absolute millivolt change and a percentage change of the original PDmax value. The maximum response to amitoride always occurred within 4 minutes. Data analysis. The PD measurements of the palm, wrist, and fingertips reflect the average of two to four independent measurements. Nasal PD is expressed as values for PDmean and PDmax. The voltage response to amiloride perfusion is expressed as both an absolute millivolt change and a percentage change of PDmax. All values, unless otherwise indicated, represent mean _+ SEM. Normal distribution of data was assured by the Kolmogorov-Smirnov test. Data were analyzed using one-way analysis of variance and the Duncan multiple-range test. Statistical significance was assumed at P <0.05. RESULTS All potentia', difference measurements were lumen negative with respect to the subcutaneous reference electrode. There was no significant difference in the nasal PDmax
values between the healthy control group ( - 2 6 + 2 mV) and the chronic disease group ( - 2 4 _+ 3 mV) (Fig. 1). The nasal PDmean values were also not significantly different between the control group ( - 1 5 + 2 mV) and the chronic disease group ( - 1 4 ___2 mV). The nasal PDmax (Fig. 1) and PDmean (Fig. 2) were both significantly different between patients with CF and non-CF controls. Measurements of PDmax or PDmean of the right versus the left nostril were not different in any of the three study groups. The nasal PDmax ( - 2 6 mV) and PDmean ( - 1 5 mV) in the one CF heterozygote studied were not different from the healthy control or chronic disease values. There was no overlap in the nasal PDmax measurements between the CF group and either the healthy control group or chronic disease group up to 2 SD from the mean (Fig. 1). Although there was no overlap of the nasal PDmean values of the CF group with either the control or chronic disease group, a single outlier from the control group fell within 2 SD of the CF mean (Fig. 2). After amiloride superfusion, nasal PD became less lumen negative in all three groups. The absolute millivolt change in nasal PD with amiloride 10-4 mol/L was significantly greater in the CF group (38 _+ 4 mV) than in either the healthy control group (10 _+ 2 mV) or the chronic disease group (9 + 1 mV). There was no significant difference in either the absolute millivott change or the percentage change of nasal PD with amiloride between the control (10 ___2 mV, 46% _+_ 5%) and chronic disease ( 9 _ 1 mV, 56% _+ 10%) groups. However, both the absolute change and the percentage change in the nasal PD values with amiloride were significantly greater in the CF group (38 ___4 mV, 70% _+ 5%) than in the non-CF groups (10 _+ 1 mY, 48% _+ 4%). There were no significant differences among the three
356
Sauder, Chesrown, and Loughlin
The Journal of Pediatrics September 1987
-I00] -801 PD
---w---*ESO
-6~
(my) -40"
. . . . . . . . . 2SD
ell
~
-20' ---4D. ....
CT
2SO
-ZSD - - - l - t -
9 --o--~ 9 9
........
9 9 9149 -2SD
CO
.........
2SD
CF
Fig. 3. Mean potential difference (PDmean, +__2 SD) for wrist. Mean value in patients with cystic fibrosis (CF) is significantly different from control (CT) and chronic disease (CD) values, but there is considerable overlap in individual data points.
T a b l e . Transepithelial potential difference
measurements at alternate sites
Palm (mV) Fingers (my) Wrist (my)
cantty lower PD in the right nostril than in the left nostril. Proper positioning of the exploring catheter to assure consistently stable nasal PD values was difficult in uncooperative patients.
Control group
Chronic disease
Cystic fibrosis
-49 _+ 3
-46 _+ 9
-54 _+ 4
DISCUSSION
-36 _+ 2
-42 +_ 6
-44 _+ 3
-26 +_ 2*
-23 _+ 5*
-41 _+ 5
Laboratory confirmation of the diagnosis of C F has been based almost exclusively on the finding of abnormal sweat electrolytes. However, accurate sweat test results are at times difficult to obtainJ 3'~7 and patients have been identified with the CF phenotype yet with borderline or normal sweat electrolyte values, 14.t6.~s Recently several investigators have demonstrated in a research setting abnormal ion fluxes in the sweat ducts 84~ and respiratory epithelia .6 in patients with CF. Knowles et al. 6.7 have demonstrated that in vivo nasal PD measurements differentiate patients with C F from the normal population and from patients with chronic disease, both in adults and in neonates. These findings suggest the need to extend this research tool into the clinical arena as a diagnostic tool. However, a previous attempt to duplicate this technique failed to confirm these results fully. ~5 Our results confirm that patients with CF have hyperpolarized nasal epithelia that have a greater voltage resl- se to superfused amiloride than do non-CF controls. These findings can be explained by an increased sodium absorption across a barrier relatively impermeable to chloride that is unique to the respiratory epithelium of patients with CF. Although the absolute values of our nasal PD measurements were not identical with those reported by Knowles et al., ~2'~ they were comparable. Our data confirm that the combination of nasal P D m a x and
Values represent mean • SEM. *P <0.05 compared with CF. study groups in the transepithelial PD measurements of the palm of the hand or between two fingertips (Table). There was a statistically significant difference in the wrist PD measurements between the C F group ( - 4 1 +_ 5 mV) and both the chronic disease ( - 2 3 + 5 mV) and control ( - 2 6 • 2 mV) groups. This difference in wrist PD measurements proved of little diagnostic benefit, however, because there was a large overlap in the wrist PD measurements of the three study groups (Fig. 3). Nasal transepithelial potential difference measurements proved limited in several situations not included in the three study groups. In two normal control patients and one patient with CF who had acute upper respiratory tract infections, and one normal control patient with acute allergic rhinitis the nasal PD was 0. In three patients (two normal controls and one with CF), abrasion of the nasal epithelia resulted in a nasal PD of 0. One C F patient who had undergone a surgical polypectomy in the right nostril 3 years previously had on two separate occasions a signifi-
Volume 111 Number 3 PDmean, coupled with the voltage response to amiloride, allowed clear differentiation between patients with C F and non-CF controls. These results suggest that in vivo nasal PD measurements may prove to b~ an additional diagnostic tool for CF. As with pilocarpine iontophoresis, t7 in vivo nasal PD measurements have several limitations. Acute upper respiratory tract infections and allergic rhinitis resulted in nasal PD measurements of 0. Acute inflammation probably causes disruption of the integrity of the epithelial tight junctions, resulting in a leaky epithelial barrier. ~9 Abnormally large passive ion fluxes across a leaky epithelial barrier could abolish the transepithelial potential difference. An alternative explanation is that metabolic inhibition may reduce active ion transport to the point where passive ion flux is equal to or greater than active ion transport. With the ability of the active ion transport mechanisms to generate an ion gradient overwhelmed, the transepithelial PD would be abolished. The exact time required to reestablish the integrity of the epithelial barrier is not known. In vivo nasal PD measurements obtained during this recovery period may result in false negative values. If in vivo nasal PD measurements are to be used in the clinical diagnosis of CF, the time course of recovery of nasal PD after an acute upper respiratory tract infection must be established. With exception of a patient who had undergone a previous polypectomy, in vivo transepithelial PD measurements were not significantly different between right and left nares. This variation in nasal PD suggests that surgical manipulation and resulting cicatrization results in altered epithelial cell types, which in turn cause falsely low nasal PD measurements. Proper positioning along the inferior turbinate was critical to obtaining accurate readings. In several instances, it was difficult to achieve proper positioning in uncooperative patients. This limitation is particularly noteworthy because many of the patients in whom the diagnosis of C F is being considered for the first time may be at an age when cooperation with a prolonged in vivo nasal PD protocol may be difficult. Unfortunately, we were unable to demonstrate other more easily accessible epithelial surfaces that would differentiate C F patients from non-CF controls. It appears that ciliated nasal epithelium is the most easily accessible surface for diagnostically useful PD measurements. In attempting to replicate the method of in vivo transepithelial PD measurements previously described by Knowles et al.,4,6,7 we made several observations that should prove useful to others interested in this technique. We believe these observations serve as the basis for the lower transepithelial PD previously reported by Hay and Geddes? 5 The standardization of the total resistance of the system and
Nasal potential difference in cystic fibrosis
357
the incorporation of a low-resistance high-impedance buffer amplifier were essential in obtaining absolute millivolt PD values comparable to those reported by Knowles et al. When home-made calomel half-cells were used and the system was used without a buffer amplifier, large variations in the resistance of the system resulted in significant current drainage and falsely low PD measurements. Although significant differences were observed in nasal PD between CF and non-CF controls without the buffer amplifier, these results differed by 50% to 75% from those previously reported by Knowles et al. 4-7and were comparable with those reported by Hay and Geddes? 5 These observations indicate the need to standardize the system used to measure in vivo PD measurements by using commercially available calomel half-cells and a highimpedance, low-resistance, calibratable buffer amplifier. In addition, each laboratory should establish its own set of reference values for known C F patients and normal controis before attempting to use in vivo nasal PD as a diagnostic tool. All of the limitations of in vivo nasal transepithelial PD measurements discussed resulted in false negative results. No false positive results were observed. Our confirmation of the findings of Knowles et al? ,6,7 suggests that in vivo nasal potential difference measurements may be useful as a diagnostic tool for CF. The complexity of obtaining in vivo nasal PD measurements and the need for meticulous attention to detail make it unlikely that in vivo nasal PD will supersede the GibsonCooke method of pilocarpine iontophoresis.2~ However, in vivo transepithelial nasal PD may prove a useful adjunct to the sweat test, especially in those patients with borderline sweat test results. We thank Donald Martin and Maxwell Stroppel for technical assistance. REFERENCES
1. Boucher RC, Bromberg PA, Gatzy JT. Airway transepithelial electric potential in vivo: species and regional differences. J Appl Physiol 1980;48:169-76. 2. Knowles MR, Carson JL, Collier AM, Gatzy JT, Boucher RC. Electric potential differences in normal human subjects in vivo. Am Rev Respir Dis 1981;124:484-90. 3. Knowles MR, Buntin W J, Bromberg PA, Gatzy JT, Boucher RC. Electric potential differences in the trachea and bronchi of human subjects in vivo. Am Rev Respir Dis 1982;126:10812. 4. Knowles MR, Gatzy JT, Boucher RC. Relative ion permeability of normal and cystic fibrosis epithelium. J Clin Invest 1983;71:1410-7. 5. Boucher RC, Knowles MR, Stutts MJ, Gatzy JT. Epithelial dysfunction in cystic fibrosis lung disease: review. Lung 1983;161:1-17. 6. Gowen CW, Lawson EE, Gingras-Leatherman J, Gatzy JT,
35$
7.
8. 9.
10.
11.
12. 13.
Sauder, Chesrown, and Loughlin
Boucher RC, Knowles MR. Increased nasal potential difference and amiloride sensitivity in neonates with cystic fibrosis. J PEDIATR 1986;108:517-21. Knowles MR, Gatzy JT, Boucher RC. Increased bioelectric potential difference across respiratory epithelia in cystic fibrosis. N Engl J Med 1981;305:1489-95. Quinton PM. Chloride impermeability in cystic fibrosis. Nature 1983;301:421-2. Quinton PM. Suggestion of an abnormal anion exchange mechanism in sweat glands of cystic fibrosis patients. Pediatr Res 1982;16:533-7. Quinton PM, Bijman J. Higher bioeleetric potentials due to decreased chloride absorption in the sweat glands of patients with cystic fibrosis. N Engl J med 1983;308:1185-9. Boucher RC, Stutts M J, Knowles MR, Cantley L, Gatzy JT. Sodium transport in cystic fibrosis respiratory epithelia: abnormal basal rate in response to adenylate cyclase activation. J Clin Invest 1986;78:1245-52. Benos DJ. Amiloride: a molecular probe of sodium transport in tissues and cells. Am J Physiol 1982;242:C131-45. Rosenstein BJ, Langbaum TS. Diagnosis. In: Taussig LM, ed. Cystic fibrosis. New York: Thieme-Stratton, 1984.
The Journal of Pediatrics September 1987 14. Anderson CM, Freeman M. Sweat test results in normal persons of different ages compared with families with fibrocystic disease of the pancreas. Arch Dis Child 1960;35:581. 15. Hay JG, Geddes DM. Transepithelial potential difference in cystic fibrosis. Thorax 1985;40:493-6. 16. Huff DS, Huang NW, Arey JB. Atypical cysti~ fibrosis of the pancreas with normal levels of sweat chloride and minimal pancreatic lesions. J PEDIATR 1979;94:237. 17. Report of the Committee for a Study for Evaluation of Testing of Cystic Fibrosis. J PEDIATR 1976;88:711-50. 18. Cogswell J J, Reidon RA, Taylor B. Suppurative lung disease in sisters mimicking cystic fibrosis. Arch Dis Child 1974; 49:520. 19. Ramphal R, Fischlschweiger W, Shands JW, Small PA. Murine influenzal tracheitis: a model for the study of influenza and tracheal epithelial repair. Am Rev Respir Dis 1979;6:1313-24. 20. Gibson LE, Cooke RE. A test for concentration of electrolyte in sweat in cystic fibrosis of the pancreas utilizing pilocarpine by iontophoresis. Pediatrics 1959;23:545-9.
We studied transepithelial potential difference (PD) in normal persons, patients with chronic disease, and patients with cystic fibrosis (CF), using the technique described by Knowles and co-workers. A maximal PD value (PDmax) and an a v e r a g e PD value (PDmean) were determined for e a c h study of the nasal respiratory epithelium. The v o l t a g e response to superfusion of 10 -4 mol/L amiloride onto nasal mucosa was noted. The PD of the palm, wrist, and between two fingertips was also measured. Nasal PDmax and PDmean of the CF group were more negative than the control (P <0.01) and chronic disease groups (P <0.01). After application of amiloride, the voltage c h a n g e in nasal PD was greater in the CF group than in the non-CF control groups (P <0.01). There were no clinically significant differences in the PD of the palm, wrist, or fingertips of the three groups. These d a t a confirm the observation that patients with CF have hyperpolarized nasal epithelia that demonstrate greater c h a n g e in response to amiloride than that in non-CF controls. These results indicate a possible role for the use of in vivo nasal PD measurements as a diagnostic test for cystic fibrosis. (J PEDIATR1987;111:353-8)
in vivo measurements of transepithelial potential difference, originally described in dog trachea by Boucher et al., 1 has recently been extended to humans by this same group. 2,3 Application of this technique to the study of cystic fibrosis has identified an abnormality in chloride transport across the epithelium of the respiratory tracP -6 and the sweat duct. 8-~~In addition to abnormal chloride transport, nasal respiratory epithelium in patients with CF Supported by American Lung Association of Florida, Pediatric Pulmonary Center Grant MCJ-2013, and New Investigation Research Award HL-26952-03 from the National Heart, Lung, and Blood Institute. Presented in part at the Annual Meeting of the Cystic Fibrosis Club, Anaheim, Calif., 1984. Submitted for publication Feb. 25, 1987; accepted May 5, 1987. Reprint requests: Gerald M. Loughlin, MD, The Johns Hopkins Hospital, Department of Pediatrics, CMSC 141, 600 N. Wolfe St., Baltimore, MD 21205.
demonstrates an increased rate of sodium absorption. 4-7'~l In vivo PD measurements in patients with C F have been used extensively by this group as a research tool, 8,12-14but these findings have not been confirmed fully in an independent clinical laboratory. Is The objectives of our study were (1) to confirm the differences in nasal PD between a group of C F patients and normal control patients or patients with CF PD
Cystic fibrosis Potential difference
[
a variety of chronic diseases, (2) to determine the difficulties and limitations of applying the technique of measuring in vivo nasal PD in a routine clinical setting, and (3) to explore other easily accessible epithelial surfaces for possible diagnostic differences between CF patients and non-CF controls.
353
354
Sauder, Chesrown, and Loughlin
The Journal of Pediatrics September 1987
-I00.
---e----
+2SD
........
-2SD
-80. ,
PDMAx
(mv)
-60' -40-
- ~-"~ ....
+2SD
9
........
+2SD
_J_k i ll ___e_____2SD
CT
It .........
CD
2SO
CF
Fig. 1. Maximum potential difference (PDmax, mean + 2 SD) for nasal epithelium in control group (CT), Patients with chronic disease (CD), and patients with cystic fibrosis (CF). Note there is no overlap in data for CF vs CD or CT groups.
METHODS Transepithelial potential difference measurements were obtained in a study population made up of 35 volunteers divided into three groups. A normal healthy control group consisted of 17 healthy nonsmokers (12 female) ranging in age from 15 to 39 years (average 26.7 years). A chronic disease control group consisted of five volunteers (three female) ranging in age from 7 to 38 years (average 20 years). The volunteers in the chronic disease group had a variety of chronic illnesses, including ulcerative colitis, rigid spine syndrome, mitral valve prolapse, reactive airway disease, and paroxysmal atrial fibrillation; two had chronic obstructive pulmonary disease with bronchiectasis of unknown cause. Sweat tests by pilocarpine iontophoresis gave normal results. Thirteen patients (10 male) known to have cystic fibrosis made up the CF group; the diagnosis of CF was confirmed by quantitative pilocarpine iontophoresis and clinical historyJ 6 The mean age of the CF group was 21 years (range 11 to 26 years). Clinical severity had no role in selecting CF patients for measurement of PD. One heterozygote, the mother of a patient known to have CF, was studied and included in the normal control group. All procedures were approved by the Institutional Review Board of the University of Florida College of Medicine. Informed consent was obtained from each patient or their parents before entering the study. Transepithelial PD measurements were obtained in a manner similar to that described in dogs and other animals by Boucher et al. H and in humans by Knowles et a13 3 PD was measured between a fluid-filled double-lumen, vinyl exploring catheter (inner diameter 0.71 mm, outer diameter 1.5 mm) (Dural Plastics & Eng PTY Ltd, Dural, New
South Wales, Australia) and a subcutaneous reference electrode. Previous studies have shown that the subcutaneous space is isoelectric with the submucosal space of airway epithelia. 14,17.18 The reference electrode was constructed by filling a sterile 23-gauge butterfly needle (Abbott Laboratories, Hospital Products Division, North Chicago, Ill.) with sterile lactated Ringer solution in 4% agar. The exploring catheter was continuously perfused with lactated Ringer solution (Travenol Labs, Inc., Deerfield, Ill.) at a rate of 0.2 to 0.4 mL/min. Both the subcutaneous reference electrode and the exploring catheter were connected by matched calomel electrodes (calomel reference electrodes, Fischer Scientific, Pittsburgh) to the input of a high-impedance, low-resistance buffer amplifier (Donald Martin, University of Florida). The output of the buffer amplifier was connected to a highimpedance digital multimeter (Dynasian Corp.) and a strip chart recorder (Moseley Autograf). PD measurements of the palm, the volar surface of the wrist, and between two fingertips were obtained by touching the fluid-filled exploring catheter to each site and recording the PD values as the average of two to four individual measurements. Nasal PD was measured along the inferior border of the inferior turbinate under direct vision using an otoscope. The exploring bridge catheter was advanced through the inferior meatus, and measurements were obtained at 0.5 cm intervals posterior to the anterior meatus, using the tip of the anterior turbinate as a landmark. An average nasal PD (PDmean) was determined for each nostril, and a total PDmean was determined for each study. A maximum nasal PD (PDmax) was recorded as the maximum stable PD measurement recorded either during entry or with-
Volume 111 Number 3
Nasal potential difference in cystic fibrosis
35S
-80"
-70-
PDMEAN
(my)
---ir---§
-60-
oo
-50-
__.LL
oo
-40-
-50-20"
-I0-
eeS ---~----.t.2S D
III 9 8 9
CT
........ 9
-2SD
--*2SD
9
-2SD
........
CD
-2SD
CF
Fig. 2. Mean potential difference (PDmean, _+ 2 SD) for nasal epithelium. Value for one patient from normal control group (CT) is within 2 SD of mean of cystic fibrosis group (CF). CD. Chronic disease group.
drawal of the exploring bridge catheter. PDmax was determined in each patient for the right and left nostrils, and for the total study. Values for baseline PD measurements were accepted if PD measurements were stable for >3 seconds and if the PD measurement at the anterior turbinate and the palm of the hand were comparable at the beginning and end of the study. After baseline PD values were obtained, the double-lumen exploring catheter was repositioned at the site of the PDmax value. Once the stable PDmax value was relocated, amiloride hydrochloride 10-4 mol/L dissolved in lactated Ringer solution was perfused at a rate of 4 ml/min for 4 minutes through the second lumen of the exploring catheter from a separate perfusion system. The change in PDmax that resulted was recorded and expressed as both an absolute millivolt change and a percentage change of the original PDmax value. The maximum response to amitoride always occurred within 4 minutes. Data analysis. The PD measurements of the palm, wrist, and fingertips reflect the average of two to four independent measurements. Nasal PD is expressed as values for PDmean and PDmax. The voltage response to amiloride perfusion is expressed as both an absolute millivolt change and a percentage change of PDmax. All values, unless otherwise indicated, represent mean _+ SEM. Normal distribution of data was assured by the Kolmogorov-Smirnov test. Data were analyzed using one-way analysis of variance and the Duncan multiple-range test. Statistical significance was assumed at P <0.05. RESULTS All potentia', difference measurements were lumen negative with respect to the subcutaneous reference electrode. There was no significant difference in the nasal PDmax
values between the healthy control group ( - 2 6 + 2 mV) and the chronic disease group ( - 2 4 _+ 3 mV) (Fig. 1). The nasal PDmean values were also not significantly different between the control group ( - 1 5 + 2 mV) and the chronic disease group ( - 1 4 ___2 mV). The nasal PDmax (Fig. 1) and PDmean (Fig. 2) were both significantly different between patients with CF and non-CF controls. Measurements of PDmax or PDmean of the right versus the left nostril were not different in any of the three study groups. The nasal PDmax ( - 2 6 mV) and PDmean ( - 1 5 mV) in the one CF heterozygote studied were not different from the healthy control or chronic disease values. There was no overlap in the nasal PDmax measurements between the CF group and either the healthy control group or chronic disease group up to 2 SD from the mean (Fig. 1). Although there was no overlap of the nasal PDmean values of the CF group with either the control or chronic disease group, a single outlier from the control group fell within 2 SD of the CF mean (Fig. 2). After amiloride superfusion, nasal PD became less lumen negative in all three groups. The absolute millivolt change in nasal PD with amiloride 10-4 mol/L was significantly greater in the CF group (38 _+ 4 mV) than in either the healthy control group (10 _+ 2 mV) or the chronic disease group (9 + 1 mV). There was no significant difference in either the absolute millivott change or the percentage change of nasal PD with amiloride between the control (10 ___2 mV, 46% _+_ 5%) and chronic disease ( 9 _ 1 mV, 56% _+ 10%) groups. However, both the absolute change and the percentage change in the nasal PD values with amiloride were significantly greater in the CF group (38 ___4 mV, 70% _+ 5%) than in the non-CF groups (10 _+ 1 mY, 48% _+ 4%). There were no significant differences among the three
356
Sauder, Chesrown, and Loughlin
The Journal of Pediatrics September 1987
-I00] -801 PD
---w---*ESO
-6~
(my) -40"
. . . . . . . . . 2SD
ell
~
-20' ---4D. ....
CT
2SO
-ZSD - - - l - t -
9 --o--~ 9 9
........
9 9 9149 -2SD
CO
.........
2SD
CF
Fig. 3. Mean potential difference (PDmean, +__2 SD) for wrist. Mean value in patients with cystic fibrosis (CF) is significantly different from control (CT) and chronic disease (CD) values, but there is considerable overlap in individual data points.
T a b l e . Transepithelial potential difference
measurements at alternate sites
Palm (mV) Fingers (my) Wrist (my)
cantty lower PD in the right nostril than in the left nostril. Proper positioning of the exploring catheter to assure consistently stable nasal PD values was difficult in uncooperative patients.
Control group
Chronic disease
Cystic fibrosis
-49 _+ 3
-46 _+ 9
-54 _+ 4
DISCUSSION
-36 _+ 2
-42 +_ 6
-44 _+ 3
-26 +_ 2*
-23 _+ 5*
-41 _+ 5
Laboratory confirmation of the diagnosis of C F has been based almost exclusively on the finding of abnormal sweat electrolytes. However, accurate sweat test results are at times difficult to obtainJ 3'~7 and patients have been identified with the CF phenotype yet with borderline or normal sweat electrolyte values, 14.t6.~s Recently several investigators have demonstrated in a research setting abnormal ion fluxes in the sweat ducts 84~ and respiratory epithelia .6 in patients with CF. Knowles et al. 6.7 have demonstrated that in vivo nasal PD measurements differentiate patients with C F from the normal population and from patients with chronic disease, both in adults and in neonates. These findings suggest the need to extend this research tool into the clinical arena as a diagnostic tool. However, a previous attempt to duplicate this technique failed to confirm these results fully. ~5 Our results confirm that patients with CF have hyperpolarized nasal epithelia that have a greater voltage resl- se to superfused amiloride than do non-CF controls. These findings can be explained by an increased sodium absorption across a barrier relatively impermeable to chloride that is unique to the respiratory epithelium of patients with CF. Although the absolute values of our nasal PD measurements were not identical with those reported by Knowles et al., ~2'~ they were comparable. Our data confirm that the combination of nasal P D m a x and
Values represent mean • SEM. *P <0.05 compared with CF. study groups in the transepithelial PD measurements of the palm of the hand or between two fingertips (Table). There was a statistically significant difference in the wrist PD measurements between the C F group ( - 4 1 +_ 5 mV) and both the chronic disease ( - 2 3 + 5 mV) and control ( - 2 6 • 2 mV) groups. This difference in wrist PD measurements proved of little diagnostic benefit, however, because there was a large overlap in the wrist PD measurements of the three study groups (Fig. 3). Nasal transepithelial potential difference measurements proved limited in several situations not included in the three study groups. In two normal control patients and one patient with CF who had acute upper respiratory tract infections, and one normal control patient with acute allergic rhinitis the nasal PD was 0. In three patients (two normal controls and one with CF), abrasion of the nasal epithelia resulted in a nasal PD of 0. One C F patient who had undergone a surgical polypectomy in the right nostril 3 years previously had on two separate occasions a signifi-
Volume 111 Number 3 PDmean, coupled with the voltage response to amiloride, allowed clear differentiation between patients with C F and non-CF controls. These results suggest that in vivo nasal PD measurements may prove to b~ an additional diagnostic tool for CF. As with pilocarpine iontophoresis, t7 in vivo nasal PD measurements have several limitations. Acute upper respiratory tract infections and allergic rhinitis resulted in nasal PD measurements of 0. Acute inflammation probably causes disruption of the integrity of the epithelial tight junctions, resulting in a leaky epithelial barrier. ~9 Abnormally large passive ion fluxes across a leaky epithelial barrier could abolish the transepithelial potential difference. An alternative explanation is that metabolic inhibition may reduce active ion transport to the point where passive ion flux is equal to or greater than active ion transport. With the ability of the active ion transport mechanisms to generate an ion gradient overwhelmed, the transepithelial PD would be abolished. The exact time required to reestablish the integrity of the epithelial barrier is not known. In vivo nasal PD measurements obtained during this recovery period may result in false negative values. If in vivo nasal PD measurements are to be used in the clinical diagnosis of CF, the time course of recovery of nasal PD after an acute upper respiratory tract infection must be established. With exception of a patient who had undergone a previous polypectomy, in vivo transepithelial PD measurements were not significantly different between right and left nares. This variation in nasal PD suggests that surgical manipulation and resulting cicatrization results in altered epithelial cell types, which in turn cause falsely low nasal PD measurements. Proper positioning along the inferior turbinate was critical to obtaining accurate readings. In several instances, it was difficult to achieve proper positioning in uncooperative patients. This limitation is particularly noteworthy because many of the patients in whom the diagnosis of C F is being considered for the first time may be at an age when cooperation with a prolonged in vivo nasal PD protocol may be difficult. Unfortunately, we were unable to demonstrate other more easily accessible epithelial surfaces that would differentiate C F patients from non-CF controls. It appears that ciliated nasal epithelium is the most easily accessible surface for diagnostically useful PD measurements. In attempting to replicate the method of in vivo transepithelial PD measurements previously described by Knowles et al.,4,6,7 we made several observations that should prove useful to others interested in this technique. We believe these observations serve as the basis for the lower transepithelial PD previously reported by Hay and Geddes? 5 The standardization of the total resistance of the system and
Nasal potential difference in cystic fibrosis
357
the incorporation of a low-resistance high-impedance buffer amplifier were essential in obtaining absolute millivolt PD values comparable to those reported by Knowles et al. When home-made calomel half-cells were used and the system was used without a buffer amplifier, large variations in the resistance of the system resulted in significant current drainage and falsely low PD measurements. Although significant differences were observed in nasal PD between CF and non-CF controls without the buffer amplifier, these results differed by 50% to 75% from those previously reported by Knowles et al. 4-7and were comparable with those reported by Hay and Geddes? 5 These observations indicate the need to standardize the system used to measure in vivo PD measurements by using commercially available calomel half-cells and a highimpedance, low-resistance, calibratable buffer amplifier. In addition, each laboratory should establish its own set of reference values for known C F patients and normal controis before attempting to use in vivo nasal PD as a diagnostic tool. All of the limitations of in vivo nasal transepithelial PD measurements discussed resulted in false negative results. No false positive results were observed. Our confirmation of the findings of Knowles et al? ,6,7 suggests that in vivo nasal potential difference measurements may be useful as a diagnostic tool for CF. The complexity of obtaining in vivo nasal PD measurements and the need for meticulous attention to detail make it unlikely that in vivo nasal PD will supersede the GibsonCooke method of pilocarpine iontophoresis.2~ However, in vivo transepithelial nasal PD may prove a useful adjunct to the sweat test, especially in those patients with borderline sweat test results. We thank Donald Martin and Maxwell Stroppel for technical assistance. REFERENCES
1. Boucher RC, Bromberg PA, Gatzy JT. Airway transepithelial electric potential in vivo: species and regional differences. J Appl Physiol 1980;48:169-76. 2. Knowles MR, Carson JL, Collier AM, Gatzy JT, Boucher RC. Electric potential differences in normal human subjects in vivo. Am Rev Respir Dis 1981;124:484-90. 3. Knowles MR, Buntin W J, Bromberg PA, Gatzy JT, Boucher RC. Electric potential differences in the trachea and bronchi of human subjects in vivo. Am Rev Respir Dis 1982;126:10812. 4. Knowles MR, Gatzy JT, Boucher RC. Relative ion permeability of normal and cystic fibrosis epithelium. J Clin Invest 1983;71:1410-7. 5. Boucher RC, Knowles MR, Stutts MJ, Gatzy JT. Epithelial dysfunction in cystic fibrosis lung disease: review. Lung 1983;161:1-17. 6. Gowen CW, Lawson EE, Gingras-Leatherman J, Gatzy JT,
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The Journal of Pediatrics September 1987 14. Anderson CM, Freeman M. Sweat test results in normal persons of different ages compared with families with fibrocystic disease of the pancreas. Arch Dis Child 1960;35:581. 15. Hay JG, Geddes DM. Transepithelial potential difference in cystic fibrosis. Thorax 1985;40:493-6. 16. Huff DS, Huang NW, Arey JB. Atypical cysti~ fibrosis of the pancreas with normal levels of sweat chloride and minimal pancreatic lesions. J PEDIATR 1979;94:237. 17. Report of the Committee for a Study for Evaluation of Testing of Cystic Fibrosis. J PEDIATR 1976;88:711-50. 18. Cogswell J J, Reidon RA, Taylor B. Suppurative lung disease in sisters mimicking cystic fibrosis. Arch Dis Child 1974; 49:520. 19. Ramphal R, Fischlschweiger W, Shands JW, Small PA. Murine influenzal tracheitis: a model for the study of influenza and tracheal epithelial repair. Am Rev Respir Dis 1979;6:1313-24. 20. Gibson LE, Cooke RE. A test for concentration of electrolyte in sweat in cystic fibrosis of the pancreas utilizing pilocarpine by iontophoresis. Pediatrics 1959;23:545-9.