Anal. Chem. 1995, 67, 2937-2942
Determination of the Inorganic Ion Composition of Rat Airway Surface Fluid by Capillary Electrophoresis: Direct Sample Injection To Allow Multiple Analyses from Nanoliter Volumes Julia C. Transfiguraoion,t Carrie Dolman,* David H. Eldelman,* and Dadd K. Lloyd*lt Division of Phamawkinetics, Depattment of Oncology, McGill Univemity, 3655 Dmmmond, Suite 701, Montreal, PO, Canada H3G 1Y6, and Meakins Christie Laboratories, McGill University, 3626 St. Utbain, Montreal, PQ, Canada, H2X 2P2
The pulmonary airways are covered by a layer of airway surface fluid (ASF) which is typically 0.14 when the peak areas for fresh and stored samples of each species were compared. Analysis of Rat ASF. Using the sampling procedures described above, ASF was collected from rats. Each sample was analyzed at least twice, once for cations and once for anions. Figure 2a shows a separation of cations in ASF from a healthy rat. Na+, K+, Mgz+, and Caz+ are clearly present in the ASF. Figure 2b shows the corresponding anion analysis. The analyses of standard samples shown in Figure 1 were taken from the calibration curve data obtained on the same day, and it can be seen that the migration times are very similar in both aqueous standards and ASF. Consistently we have measured large C1concentrationsin rat ASF but no significant concentrationsof other high mobility anionic species. The concentrations determined in four rats, sampled on the same day, are shown in Table 1. In each rat, the sample was taken from the same location, 2 cm below the opening of the trachea. We investigated collection of ASF at various positions in the airway and found that the most consistent results were obtained at this position. The sampling position may have to be varied when other strains of rats or other species are used. When possible, duplicate samples were taken and analyzed from each site, one after the other. These measurements consistently show that rat ASF is hypotonic. Na+ concentrations were very consistent, with a mean value of 43.5 f 2.2 mM. C1was present at lower concentrations than Na+ in all but one sample, with a mean value of 35 f 8 mM. The other cations were present at lower levels: K+, 1.6 f 0.3 mM; Mg2+,0.5 f 0.07 mM; Ca2+,0.4 f 0.09 mM. Although there is a higher variability associated with the anion analyses, this is unlikely to account for
Tabls 2. Duplicate Inorganic Ion Determinations from the Same Sample'
a. cation analvsis
ion concn (mM)
rat
Na+
K+
Mg2+
Ca2+
c1-
A
41, 46 42,45 46,42
2.2, 1.6 1.6, 1.9 2.1, 1.9
0.6, 0.6 0.4,0.5 0.3,0.3
0.3, 0.4 0.4,0.4 0.7, 1.0
39, 42 29, 29 47, 42
B E
Rats A and B are the same as A and B in Table 1;E was analyzed in a different set of experiments. 0
1
2
3
4
5
6
7
timelmin
b. anion analysis
0
1
I
3
2
5
4
7
6
timelmin
Figure 2. (a) Separation of cations in rat ASF: (1) K+, (2) Caz+, (3) Na+, and (4) Mg2+. The inset shows the the electropherogram from 4 to 7 min magnified five times in the vertical axis. (b) Anion analysis of rat ASF: (5) CI-. Separation conditions as in text. No other anions have as yet been detected in rat ASF using the present method. The concentrationsdetermined for this sample are reported for A1 in Table 1. Both analyses were made from a total volume of -100 nL of ASF collected in one sampling capillary. Table 1. lnorganlc ion Concentrations Determined In Four Brown-Norway Rats'
ion concn (mM) rat
sample no.
Naf
K+
Mg2+
Ca2+
C1-
A
1 2 1 2 1 1
41 46 42 45 46 41
2.2 1.6 1.6 1.9 1.2 1.3
0.6 0.6 0.4 0.5 0.5 0.5
0.3 0.4 0.4 0.4 0.6 0.5
39 51 33 31 30 28
B C D
a Sampling 2 cm below the larynx. Samples were collected for 2 min each, with a second sample being taken immediately after the first. For rats C and D, the amount of fluid collected on the second sampling was too small for analysis.
the wide range of measured C1- values, which seems to reflect a large variability in the concentration of this ion in rat ASF. To determine both cation and anion concentrations, it is necessary to perform two injections from each sample, and most samples collected in healthy rats can easily be analyzed in this way. If the volume of ASF collected is very small (e.g., ~ 0 . mm 5 of the sampling capillary filled), even one injection might not be possible with the manual injection technique we are using at present, although use of a micromanipulator for positioning the two capillaries for injection may allow some improvements. If a relatively large quantity of ASF is collected (e.g., 2 2 mm of
sampling capillary filled), multiple analyses can be made. Although we do not routinely do this for each sample, some data on duplicate cation and anion analyses from single rat ASF samples are shown in Table 2. These show reasonable agreement in all cases. Since we know of no other way of directly analyzing ASF in rats, the method could not be cross-validated against another technique. However, the inorganic ion concentrations in rat ASF presented here can be compared with those determined in previous studies of ASF in large animals and humans. Some of these previous results have been quite variable; ASF in canine trachea was found to be isotonic by Boucher et al.,5but Connoley et al.'j found reduced Na+ and increased K+ levels. ASF in noninfected human trachea was reported to be hypotonic by Joris and Quinton? More recent in vivo measurements confirm the hypotonicity of ASF in healthy human airways and also the presence of significant K+ concentrations, -29 mM; in diseased airways, the Na+ and C1- concentrations were observed to increase? To overcome the technical difficulty in sampling from small airways in vivo, Joris and Quinton studied ASF in vitro* by isolating small airways derived from lung tissue after surgery; no signifcant differences were observed from the measurements made by the same group of ASF in the trachea. The present results clearly indicate that rat ASF is hypotonic, which is similar to measurements made in humans? However, unlike human ASF, there is very little K+ in the rat ASF. The sampling and analysis methods described in this article will allow us to develop a new understanding of the relation between ASF composition and function. Although previously ASF sampling using filter paper has been possible in humans or larger animals, this technique could not be applied to rats or mice. CE is very flexible in terms of the variety of compounds that can be analyzed, and in future, we will be looking at other components of ASF. Other compounds may also be analyzed by bronchoalveolar lavage, but lavage gives an overall picture of the lung fluid composition, while the method described here allows sampling from discrete sites. Furthermore, lavage experiments generally provide only qualitative data on ASF composition (or at best semiquantitative determination of ASF components using urea as a marker of dilutionz1). We have shown that the sampling method described here is capable of providing quantitative analyses with RSDs of -10% or less, which is acceptable for most bioanalytical procedures, and there is no reason to expect worse performance for the determination of other ASF components since CE has a proven record in the direct analysis of biofluids.18 In addition, CE equipment is quite widely available, probably more so than (21) Rennard, S.;Basset, G.; Lecossier, D.; O'Donnell, IC;Martin,P.; Pinkston, P.; Crystal, R G. J. Appl. Physiol. 1986, 60,532-538.
Analytical Chemistry, Vol. 67, No. 17, September 1, 1995
2941
the X-ray fluorescence instrumentation used in previous studies of inorganic ions in the ASF of large mammals.
recipients of chercheur-boursier awards from the Fonds de la Recherche en Santb du QuCbec.
ACKNOWLEWMENT
Received for review February 9,1995. Accepted May 25,
Supported by the Canadian Cystic Fibrosis Research Foundation, the EL/JTC Costello Foundation and the Respiratory Health Network of Centres of Excellence. D.K.L. and D.H.E.were
AC9501506
2942 Analytical Chemistry, Vol. 67, No. 17, September 7, 7995
1995.B @Abstractpublished in Advance ACS Absfrucfs,July 1, 1995.
