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Determination of amino acids in polymers with varying structure and charge was performed using vapor phase acid hydrolysis and subsequent precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). Percent load of neutral, cationic and anionic peptide-modified synthetic polymers was accurately determined using this technique. Assay utility was shown for glycosaminoglycans and other sulfated polymers, neutral carbohydrate polymers such as agar, agarose, and cellulose, and polymers such as lipopolysaccharide and deoxyribonucleic acid. The carboxylated and sulfated molecules included chondroitin sulfate, hyaluronic acid, dermatan sulfate, and heparin, and the sulfated polymers included fucoidan, carrageenan, and dextran sulfate, as examples. Assayed cumulative amino acid concentrations (i.e. protein levels) are reported, although amino acid distribution data was also available from the analysis. Recovery was acceptable for the various compounds tested and did not correlate with structure. However, different sample sizes were necessary to achieve acceptable recovery, depending on the level of protein present in the matrix. While some matrices contained peaks in addition to the amino acids and amino sugars, they were not found to interfere using the standard gradient separation. Assayed amino acid profiles were compared for agaroses with differing electroendosmosis values and for agar samples from different parts of the globe. While the amounts of protein varied depending on source, the relative distribution of amino acids was very similar across the agar samples surveyed.

Human airways are kept sterile by a mucosal innate defense system that includes mucus secretion. Mucus is secreted in healthy upper airways primarily by submucosal glands and consists of defense molecules mixed with mucins, electrolytes, and water and is also a major component of sputum. Mucus traps pathogens and mechanically removes them via mucociliary clearance while inhibiting their growth via molecular (e.g., lysozyme) and cellular (e.g., neutrophils, macrophages) defenses. Fluid secretion rates of single glands in response to various mediators can be measured by trapping the primary gland mucus secretions in an oil layer, where they form spherical bubbles that can be optically measured at any desired interval to provide detailed temporal analysis of secretion rates. The composition and properties of the mucus (e.g., solids, viscosity, pH) can also be determined. These methods have now been applied to mice, ferrets, cats, pigs, sheep, and humans, with a main goal of comparing gland secretion in control and CFTR-deficient humans and animals.

Human airways are kept sterile by a mucosal innate defense system that includes mucus secretion. Mucus is secreted in healthy upper airways primarily by submucosal glands and consists of defense molecules mixed with mucins, electrolytes, and water and is also a major component of sputum. Mucus traps pathogens and mechanically removes them via mucociliary clearance while inhibiting their growth via molecular (e.g., lysozyme) and cellular (e.g., neutrophils, macrophages) defenses. Fluid secretion rates of single glands in response to various mediators can be measured by trapping the primary gland mucus secretions in an oil layer, where they form spherical bubbles that can be optically measured at any desired interval to provide detailed temporal analysis of secretion rates. The composition and properties of the mucus (e.g., solids, viscosity, pH) can also be determined. These methods have now been applied to mice, ferrets, cats, pigs, sheep, and humans, with a main goal of comparing gland secretion in control and CFTR-deficient humans and animals.

To assess CFTR function in vivo, we developed a bioassay that monitors and compares CFTR-dependent and CFTR-independent sweat secretion in parallel for multiple (~50) individual, identified glands in each subject. Sweating was stimulated by intradermally injected agonists and quantified by optically measuring spherical sweat bubbles in an oil-layer that contained dispersed, water soluble dye particles that partitioned into the sweat bubbles, making them highly visible. CFTR-independent secretion (M-sweat) was stimulated with methacholine, which binds to muscarinic receptors and elevates cytosolic calcium. CFTR-dependent secretion (C-sweat) was stimulated with a β-adrenergic cocktail that elevates cytosolic cAMP while blocking muscarinic receptors. A C-sweat/M-sweat ratio was determined on a gland-by-gland basis to compensate for differences unrelated to CFTR function, such as gland size. The average ratio provides an approximately linear readout of CFTR function: the heterozygote ratio is ~0.5 the control ratio and for CF subjects the ratio is zero. During assay development, we measured C/M ratios in 6 healthy controls, 4 CF heterozygotes, 18 CF subjects and 4 subjects with 'CFTR-related' conditions. The assay discriminated all groups clearly. It also revealed consistent differences in the C/M ratio among subjects within groups. We hypothesize that these differences reflect, at least in part, levels of CFTR expression, which are known to vary widely. When C-sweat rates become very low the C/M ratio also tended to decrease; we hypothesize that this nonlinearity reflects ductal fluid absorption. We also discovered that M-sweating potentiates the subsequent C-sweat response. We then used potentiation as a surrogate for drugs that can increase CFTR-dependent secretion. This bioassay provides an additional method for assessing CFTR function in vivo, and is well suited for within-subject tests of systemic, CFTR-directed therapeutics.