The sweat chloride test remains the gold standard for confirmatory diagnosis of 
cystic fibrosis (CF) in support of universal newborn screening programs. 
However, it provides ambiguous results for intermediate sweat chloride cases 
while not reflecting disease progression when classifying the complex CF disease 
spectrum given the pleiotropic effects of gene modifiers and environment. Herein 
we report the first characterization of the sweat metabolome from 
screen-positive CF infants and identify metabolites associated with disease 
status that complement sweat chloride testing. Pilocarpine-stimulated sweat 
specimens were collected independently from two CF clinics, including 50 
unaffected infants (e.g., carriers) and 18 confirmed CF cases. Nontargeted 
metabolite profiling was performed using multisegment injection-capillary 
electrophoresis-mass spectrometry as a high throughput platform for analysis of 
polar/ionic metabolites in volume-restricted sweat samples. Amino acids, organic 
acids, amino acid derivatives, dipeptides, purine derivatives, and unknown 
exogenous compounds were identified in sweat when using high resolution tandem 
mass spectrometry, including metabolites associated with affected yet 
asymptomatic CF infants, such as asparagine and glutamine. Unexpectedly, a 
metabolite of pilocarpine, used to stimulate sweat secretion, pilocarpic acid, 
and a plasticizer metabolite from environmental exposure, 
mono(2-ethylhexyl)phthalic acid, were secreted in the sweat of CF infants at 
significantly lower concentrations relative to unaffected CF screen-positive 
controls. These results indicated a deficiency in human paraoxonase, an enzyme 
unrelated to mutations to the cystic fibrosis transmembrane conductance 
regulator (CFTR) and impaired chloride transport, which is a nonspecific 
arylesterase/lactonase known to mediate inflammation, bacterial biofilm 
formation, and recurrent lung infections in affected CF children later in life. 
This work sheds new light into the underlying mechanisms of CF pathophysiology 
as required for new advances in precision medicine of orphan diseases that 
benefit from early detection and intervention, including new molecular targets 
for therapeutic intervention.