The sweat chloride test was introduced nearly 60 years ago and still represents the gold standard for diagnosing cystic fibrosis (CF) in infants who have tested positive in screening programs. Elevated levels of chloride in sweat (≥60 mM) are considered confirmatory for CF, but the sweat test can be somewhat ambiguous when chloride levels are intermediate (30–59 mM), and this can complicate clinical decision making. A positive sweat chloride result also isn’t predictive of how severely cystic fibrosis might develop in any one individual with a specific mutation in the causative cystic fibrosis transmembrane conductance regulator (CFTR) gene.

Scientists at McMaster University in Ontario, Canada, have now identified two new biological markers of CF that could help to improve diagnosis and provide new insights into mechanisms of the disease. Both of the markers found in the sweat of infant CF patients are exogenous compounds, which means that neither is a native metabolite from cellular activity or from dietary nutrients. Rather, one of the two identified compounds is a drug metabolite, and the other is the metabolite of a chemical to which we are just about all exposed environmentally.

“… there has there has been a recent resurgence in the interest in sweat analysis as it provides a simple and noninvasive way to evaluate the health status of an individual,” lead investigator Philip Britz-McKibbin, Ph.D., associate professor at the University’s department of chemistry and chemical biology, told GEN. “There have been other studies published to evaluate the potential of sweat as a biofluid for performing clinical diagnostics or drug testing—although it is still not routine especially in terms of methods used for standardized and reproducible sweat collection. …Our motivation was first curiosity since there was little information on the exact chemical composition of sweat, especially from infants within a clinical setting, in this case screen-positive CF infants.”

The team’s second motivator was those “diagnostic dilemmas” that result from the 10% of screen positives that generate borderline sweat chloride results. “This creates a lot of stress for families and physicians as it is difficult to evaluate whether the infant has CF (CF is a really a disease spectrum), or may develop CF later in life (perhaps as a late onset or mild phenotype).” Less commonly, a small fraction of screen-positive CF infants exhibit normal sweat chloride results (<30 mM), but may develop some CF-like symptoms later in life. These cases are referred to as atypical or nonclassic forms of CF.

The McMaster University researchers have developed a high-throughput, nontargeted metabolite profiling technology, known as multisegment injection-capillary electrophoresis-mass spectrometry (or MSI-CE-MS), for analyzing polar/ionic metabolites. The versatile MSI-CE-MS technology is “ideal for analysis of ‘volume-restricted’ biospecimens, such as the 2–5 μL of residual sweat from infants,”  professor Britz-McKibbin noted. The technique was applied to characterize the sweat metabolome of 18 affected and confirmed infant CF cases and another 50 unaffected screen-positive infants, primarily carriers who have a single disease-causing CFTR mutation that is picked up during newborn screening. Samples were collected over a two-year period from CF clinics at the McMaster Children’s Hospital and The Hospital for Sick Children in Toronto.

“Our main goal was to characterize the sweat metabolome from screen-positive CF infants, and then demonstrate that other small molecules (once detected, identified, and shown to be significant) can also distinguish CF disease status in largely asymptomatic infants early in life (typically about 3 to 4 weeks old) beyond sweat chloride or other major electrolytes, such as sodium which have been reported in past,” GEN was told.

The MSI-CE-MS analysis picked out numerous compounds in the infants' sweat samples. “Sweat metabolites detected consistently in screen-positive CF infants comprised a diverse range of compounds, including amino acids, dipeptides, organic acids, fatty acids, and several exogenous chemicals, such as paraben-based preservatives,” the authors write in their published paper in ACS Central Science, which is entitled “The Sweat Metabolome of Screen-Positive Cystic Fibrosis Infants: Revealing Mechanisms beyond Impaired Chloride Transport.”

More specifically, the analysis identified two compounds that were secreted in the sweat of CF infants at much lower concentrations than in the sweat of the CF screen-positive controls. Unexpectedly, the compound that was found to be most significant for distinguishing CF from non-CF-affected infants was pilocarpic acid, a metabolite of pilocarpine, the drug that is used to stimulate the sweat response. “In other words, the infant's response to the drug used to stimulate sweat indicated a distinct difference in their disease status,” Dr. Britz-McKibbin noted to GEN.”

The second exogenous compound linked with CF was identified as monoethylhexylphthalate (MEHP), a metabolite of the ubiquitous plasticizer diethylhexylphthalate ester, or DEHP, to which we are all exposed as it is used widely in plastics, household items, and other devices. “Infants are likely exposed upon breastfeeding and prenatally due to maternal exposure,” he suggested.

Neither of the two exogenous compounds identified using the MSI-CE-MS technique has been associated with CF before, Britz-McKibbin stressed. “The two unexpected metabolites inferred that CF infants had a underlying deficiency in an unrelated protein to CFTR  namely paraoxanase, an enzyme that has many roles to hydrolyze lipids and toxicants, including organochlorine pesticides.”

Identification of the pilocarpine metabolite and MEHP as biological markers for CF led the team to formulate the hypothesis that human paraoxanase may be associated with the CF disease spectrum in parallel with primary mutations in the CFTR gene. 

It’s not the first time that paraoxanase has been associated with CF, Britz-McKibbin pointed out. Gene association studies carried out in the mid-1980s had linked the enzyme with CF even before the discovery of CFTR.  “Also, the enzyme is known to mediate inflammation and bacterial biofilm in older CF patients since its function is to hydrolyze ‘quorum-sensing' molecules from Pseudomonas in lungs. A deficiency in paraoxonase thus renders CF patients more prone to recurrent lung infections in addition to impairments in chloride transport mediated by CFTR.”

The identification of proteins associated with the CF disease spectrum could offer new targets for drug therapy, Dr. Britz-McKibbin indicated to GEN. “Thus, paraoxonase deficiency could be treated with small molecules that activate the enzyme, which could be a valuable adjuvant therapy to enhance the efficacy of antibiotic treatment against recurrent lung infections.”

The McMaster University researchers plan to further validate the findings in a larger cohort of CF infants and evaluate whether the sweat metabolome could offer additional insights into disease progression and response to novel therapies, including potentiators such as ivacaftor, which are designed to restore the function of the mutant CFTR for certain genotypes, Dr. Britz-McKibbin continued. Having already protected their IP, the team is also considering potential partnerships with industry to translate the basic research into clinically relevant R&D. “In short, there are still opportunities to further improve CF screening (reduce identification of unaffected carriers who do not have CF) as well as prognostic indicators (predict who later develops CF, especially for initial borderline sweat cases) and treatment responsivity (new therapies for CF, including other protein targets, such as CFTR).”