Serum Polyfluoroalkyl Concentrations, Asthma Outcomes, and Immunological Markers in a Case–Control Study of Taiwanese Children

Background: Perﬂuorinated compounds (PFCs) are ubiquitous pollutants. Experimental data suggest that they may be associated with adverse health outcomes, including asthma. However, there is little supporting epidemiological evidence. Methods: A total of 231 asthmatic children and 225 nonasthmatic controls, all from northern Taiwan, were recruited in the Genetic and Biomarkers study for Childhood Asthma. Structure questionnaires were administered by face-to-face interview. Serum concentrations of 11 PFCs and levels of immunological markers were also measured. Associations of PFC quartiles with concentrations of immunological markers and asthma outcomes were estimated using multivariable regression models. Results: Nine PFCs were detectable in most children (≥ 84.4%), of which perfluorooctane sulfonate (PFOS) was the most abundant (median serum concentrations of 33.9 ng/mL in asthmatics and 28.9 ng/mL in controls). Adjusted odds ratios for asthma among those with the highest versus lowest quartile of PFC exposure ranged from 1.81 (95% CI: 1.02, 3.23) for the perfluorododecanoic acid (PFDoA) to 4.05 (95% CI: 2.21, 7.42) for perfluorooctanic acid (PFOA). PFOS, PFOA, and subsets of the other PFCs were positively associated with serum IgE concentrations, absolute eosinophil counts (AEC), eosinophilic cationic protein (ECP) concentrations, and asthma severity scores among asthmatics. Conclusions: This study suggests an association between PFC exposure and juvenile asthma. Because of widespread exposure to these chemicals, these findings may be of potential public health concern.

Milli-Q water was cleaned using Waters Oasis HLB Plus (225 mg) cartridges (Milford, MA) to remove the potential residue of PFCs. Mixed stock PFC standard solution was prepared in methanol. All reagents were used as received.

SAMPLE PREPARATION AND EXTRACTION
Serum samples were extracted following a method developed by Hansen et al. (2001) . Two millilitres of 0.25 M Na 2 CO 3 and one liter of 0.5 M TBAHS were added to 0.5 mL of serum and then extracted twice with MTBE. The combined MTBE extracts were brought to dryness under a gentle stream of high purity nitrogen, and reconstituted in 1 mL mixture of methanol and 10 mM ammonium acetate (2:3, v/v) before final filtration with a 0.22 μm nylon filter.

INSTRUMENTAL ANALYSIS
Extracts of serum samples were analyzed via high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Chromatography was performed by an Agilent 1200 HPLC system (Palo Alto, CA). A 25 μL aliquot of extract was injected onto a 2.1 × 100 mm (3.5 μm) Agilent Eclipse Plus C18 column (Palo Alto, CA) with 10 mM ammonium acetate and acetonitrile as mobile phases starting with 40% acetonitrile at a flow rate of 250 μL/min and column temperature of 40 °C. The gradient was increased to 90% acetonitrile at 9 min and then held for 2 min. In addition, an 8 min re-equilibration interval was run before each following sample. The HPLC system was interfaced to an Agilent 6410 Triple Quadrupole (QQQ) mass spectrometer (Santa Clara, CA) operated with electrospray ionization (ESI) in negative mode.
Instrumental parameters were optimized to transmit the [M-K]ion before fragmentation to one or more product ions. Declustering potential and collision energies were optimized for each analyte and ranged from 35 to 90V and 10 to 35eV, respectively. Data were acquired by tandem mass spectrometry using multiple reaction monitoring ( Moreover, multiple daughter ions were monitored for confirmation, but quantitation was based on a single production. In all cases, the capillary was held at -4 kV and the desolvation temperature was kept at 350 °C.

QUALITY ASSURANCE AND QUALITY CONTROL
Procedural blanks were prepared at an interval of every ten samples to check if contamination had occurred during the extraction of samples. Solvent blanks containing acetonitrile and Milli-Q water (2:3, v/v) were run after every twenty samples to monitor for background contamination.
Duplicate injections and calibration check standards were run after every twenty samples to assure the precision and accuracy of each run. Matrix spike recoveries were tested by spiking native standards of all 10 target compounds into 12 randomly selected samples, at levels of 10 ng and 20 ng for each of the target compounds. All the matrix spike samples were analyzed in duplicate.
Recoveries of native standards spiked in serum matrix were 98 ± 5%, 101 ± 6%, 95 ± 3%, and 96 ± 5%, for PFOS, PFOA, PFHxS, and PFNA, respectively. The average recoveries for other perfluorochemicals ranged from 82% to 96%. The relative standard deviations (RSD) of duplicate analyses were less than 5% for PFOS, PFOA, PFBS, PFDA, PFHpA, PFHxA, PFHxS, PFNA, PFTA,and less than 10% for PFDoA. The concentrations of serum extracts were quantified via nine-point matrix-matched calibration curves ranging from 0.01 to 100 ng/mL, which were performed by adding mixed PFC standard solution into blank and newborn bovine serum, respectively. The regression coefficients (r 2 ) of calibration curves for all the target analytes in different matrixes were higher than 0.99. The limit of detection (LOD) was defined as the peak of analyte that needed to yield a signal-to-noise (S/N) ratio of 3:1, and the limit of quantification (LOQ) was defined as the lowest point on the standard curve, above the LOD, with a RSD less than 10%.