Methods for determining the composition of nasal fluid by X‐ray microanalysis

Abstract

The nasal fluid is an easily accessible form of airway surface liquid. The objective of this study was to find a technically easy and reproducible method for sampling and analysis of this fluid. In a pilot study, several methods to carry out X-ray microanalysis of sub-microliter droplets were compared. Acceptable results were obtained with several of these methods (pipeting on filter paper or analysis of frozen-hydrated droplets at low temperature). Nasal fluid was collected from the inferior turbinate with a micropipette after occlusion of a nostril for 5-10 minutes. Ion concentrations in nasal fluid from six control subjects were (in mM, mean +/- standard error): sodium (Na) 127 +/- 6, chloride (Cl) 140 +/- 7, potassium (K) 27 +/- 3, and calcium (Ca) 5 +/- 1. This sampling method proved difficult to apply to cystic fibrosis (CF) patients because of the viscous quality of their nasal secretion. Therefore, an alternative method was devised. Sephadex G-25, ion exchange beads were mounted on double-sided tape, which was stuck on a filter paper as support. The filter paper was applied for 10 minutes to the nostril of a subject, and kept loosely in place. During the exposure period, the nasal fluid equilibrates with the beads. After removal of the filter paper with the beads from the nostril, the beads were rinsed with a hydrophobic volatile silicone oil to remove excess nasal fluid, dried, and analyzed. This method of collection is not cumbersome for the subject and gives results similar to those obtained by the direct collection method: Na 142 +/- 28 mM, Cl 150 +/- 36 mM, K 43 +/- 10 mM (mean and standard error of four determinations). Small differences between the filter method and the bead method can be explained by the fact that the filter method measured total nasal fluid, whereas the bead method measures predominantly the fluid component. Subjects suffering from mild respiratory illness or rhinitis had higher values for Na, K, and Cl in their nasal fluid.

X-ray microanalysis of airway surface liquid in the mouse

“…In the frozen-hydrated trachea, the entire ASL is sampled, with probably a predominance of the upper mucous layer. Airway mucus has a composition that is different from that of the watery fraction (20). Apart from mucus macromolecules contributing S, this layer also contains cell debris that would contribute elements such as P and K. Leakage from dead cells could partially explain the relatively high K concentration in the watery fraction.…”

“…The needle was connected to a syringe previously filled with air, and the beads were sprayed evenly over the tracheal surface by pressure on the syringe. Earlier experiments have shown that saturation of the beads with a salt solution is obtained after 5 min (20). After absorption of the ASL, the beads were recovered by being flushed with hydrophobic volatile silicone oil (Dow Corning 200/1cS; BDH, Poole, UK) and collected in a watch glass (12).…”

“…The Sephadex G-25 beads were applied to double-sided tape (3M, Minneapolis, MN) attached to a filter paper (width 1-2 mm, length 5 mm; Schleicher and Schuell, Dassel, Germany) (20). The filter paper with the beads was inserted into one or both nostrils of the mouse, with beads facing the nasal septum, and were kept there for 10 min.…”

“…Typically, 10 -12 beads were analyzed from each sample. For quantitative analysis, the data were compared with the results obtained on beads soaked in salt solutions of different concentrations (50 -250 mM) (20).…”

Measurements of airway surface liquid height and mucus transport by fluorescence microscopy, and of ion composition by X-ray microanalysis

“…Sephadex is the trade name for a cross-linked dextran gel with ion-exchange capacity, and these beads saturate with salt solution within about 5 min [19]. The beads may be sprayed on to a sample via a syringe, or mounted on a filter paper (see Section 9) and it is important that the beads are well spread and do not stick together.…”