General characteristics of participants are reported in . The average age was 11.1±4.5 years (mean± standard deviation) and participation was similar across genders (49% girls and 51% boys). Of the 12,476 children included in the current study, >10,000 (>80%) had perfluoroalkyl acid quantification and serum lipids available for analysis. Consistent with Census Bureau estimates for the area, more than 95% reported their ethnicity as “White”, almost 40% (39.7%) were classified as overweight or obese (≥85th BMI percentile), and approximately one-third (36.7%) reported having a regular exercise program. Based on participant-reported residence at the time of their enrollment, a slightly higher proportion of participants were from Ohio compared to West Virginia (54% vs. 44%, respectively).
Mean total-C was 160.7±29.3 mg/dL, with 65.8% classified as acceptable (<170 mg/dL). Mean calculated LDL-C was 87.3±25.2 mg/dL, with 83.7% classified as acceptable (<110 mg/dL). Mean HDL-C was 49.3±11.3 mg/dL, with 92.2% classified as ideal (≥35 mg/dL) and 19.7% <40 mg/dL. Finally, mean fasting triglycerides was 99.1±56.0 mg/dL, with 85.7% classified as acceptable (≤150 mg/dL).
Mean serum PFOA and PFOS concentration was 69.2±111.9 ng/mL and 22.7±12.6 ng/mL respectively, with serum concentrations statistically significantly higher in males and younger children for both PFOA and PFOS, particularly PFOA. Consistent with the environmental (drinking water) contamination and as previously reported,15
serum PFOA concentration for 12–19 year olds in the Project population was substantially above the reported concentration for 12–19 year olds in the 2003–2004 NHANES study,5
while PFOS concentrations were similar (29.3 ng/mL vs. 3.9 ng/mL and 19.1 ng/mL vs 19.3 ng/mL respectively).
Results from regression analysis (not shown) demonstrated that, after adjustment for covariables, total-C and LDL-C were linearly and positively associated with both PFOA and PFOS (p<0.0001 for all models). Triglycerides were also linearly and positively associated with PFOA (p=0.019) but not PFOS. HDL-C was not linearly associated with PFOA but was positively associated with PFOS. For linear regression models and other analyses reported here, it was almost always the case that all covariables were also statistically significantly associated with the dependent variable (not shown).
Associations between increasing PFOA and PFOS quintiles and the covariable-adjusted EMM of serum lipids are depicted in . Both total-C and LDL-C demonstrated a consistent increase for each increase in PFOA or PFOS quintile: a 4.6 mg/dL and 3.8 mg/dL increase in the covariable-adjusted EMM of total-C and LDL-C between the 1st and 5th quintile of PFOA; and a 8.5 mg/dL and 5.8 mg/dL increase in the covariable-adjusted EMM of total-C and LDL-C between the 1st and 5th quintile of PFOS. Overall associations between PFOA and PFOS and HDL-C and triglycerides were less clear with no apparent association between PFOA quintile and covariable-adjusted EMM for HDL-C, or PFOS quintile and covariable-adjusted EMM for triglycerides. There was a small increase in the covariable-adjusted EMM of HDL-C and the 1st and 3rd quintile of PFOS, but not the 3rd to 5th quintile of PFOS. There was no difference between the covariable-adjusted EMM for triglycerides and the 1st, 2nd, or 3rd PFOA quintile, but an increase between the 3rd and 4th or 5th quintile of PFOA.
Changes in Covariate-Adjusted Estimated Marginal Means (GLM Analysis) Across PFOA and PFOS Quintiles
These associations are more fully characterized in , which reports differences between the 5th–1st quintile and β-for-trend for age group and gender strata. For PFOA and total-C and LDL-C, there was a trend toward a larger increase in the covariable-adjusted EMM in younger compared to older age groups (5.8 mg/dL vs. 4.2 mg/dL for total-C and 4.92 mg/dL vs. 3.24 mg/dL for LDL-C), and in boys compared to girls (within each age group). The β-for-trend for each of these age group and gender strata were all statistically significant (p<0.05). For PFOS, there was a trend toward a larger increase in the covariable-adjusted EMM in older compared to younger age groups (9.5 mg/dL vs. 5.5 mg/dL for total-C and 7.5 mg/dL vs. 3.4 mg/dL for LDL-C), and a trend toward larger increases for boys compared to girls (within each age group). The β-for-trend for each of these age group and gender strata were also all statistically significant (p<0.05).
Differences in PFOA between 1st–5th Quintile Estimated Marginal Means (GLM Analysis) and Assessment of Quintile Trend (Regression Analysis)
In contrast, the β-for-trend for age group and gender strata for the associations between PFOA and HDL-C and triglycerides were not statistically significant (except for one stratum), making differences in the covariable-adjusted EMM difficult to interpret. For associations between PFOS and HDL-C, the β-for-trend was statistically significant for boy and both genders combined (but not girls) for both age groups, though marginal increases in covariable-adjusted EMM were small (1.1 mg/dL to 2.6 mg/dL). Similarly, the β-for-trend for age group and gender strata for the associations between PFOS and triglycerides were not statistically significant (except for one stratum), making differences in the covariable-adjusted EMM difficult to interpret.
In and , the covariable-adjusted EMM for serum lipids for 20-group quantiles of PFOA or PFOS are plotted against the median of PFOA or PFOS for the quantile. For both PFOA and PFOS, the results demonstrated a non-linear association between increasing PFOA or PFOS and total-C and LDL-C. For PFOA, the largest increases in the covariable-adjusted EMM of total-C and LDL-C were seen at the lowest range of PFOA concentrations; the slope attenuated at higher serum concentrations. Although population exposure and corresponding serum concentrations of PFOS were lower, the relationship between increasing PFOS quantiles and the covariable-adjusted EMM of total-C and LDL-C was similar across the spectrum of serum PFOS concentrations. HDL-C also demonstrated a small, non-linear association with increasing PFOS.
Non-Linear Changes in Covariate-Adjusted Estimated Marginal Means (GLM Analysis) Across PFOA 20-Group Quantiles
Non-Linear Changes in Covariate-Adjusted Estimated Marginal Means (GLM Analysis) Across PFOS 20-Group Quantiles
Logistic regression results are shown in . Both increasing PFOA and PFOS quintiles were positively associated with an increased risk of abnormal total-C (adjusted odds ratio 1.2 (95% CI 1.1–1.4) and 1.6 (1.4–1.9) respectively) and LDL-C (1.4 (1.2–1.7) and 1.6 (1.3–1.9) respectively); increasing PFOS quintiles were also associated with decreased risk of low HDL-C (0.7 (0.6–0.9)). Neither PFOA nor PFOS were associated with increased risk for abnormal triglycerides.
Risk of Abnormal Blood Lipids (Logistic Regression Analysis) Based on Increasing PFOA & PFOS Quintiles
Results for analyses assessing interaction are shown in . For total-C, LDL-C and triglycerides, results do not support interaction between PFOA and PFOS in the prediction of these lipids. There is some evidence of multiplicative interaction between PFOA and PFOS in the reduction of the risk of low HDL-C (the odds ratio for Group 4 exceeds the product of the odds ratios for Group 2 and Group 3).
Assessment of Interaction (Logistic Regression Analysis) Coincident PFOA & PFOS Quintile Groups
Sensitivity analyses were conducted to assess the stability of the association between PFOA or PFOS and lipids after the inclusion of household income and for fasting-only participants. Results (not shown) suggested that, after adjustment for the same covariables, models were stable and associations unaltered. The positive and statistically significant association between both PFOA and PFOS and total-C and LDL-C was not altered after the inclusion of household income, a proxy for socioeconomic status, or when analysis was performed only on the subset of participants who had completed a ≥6 hr fast. Likewise, the positive, statistically significant association between PFOA and fasting triglycerides was not altered after the inclusion of household income. PFOS was not associated with fasting triglycerides with or without the inclusion of household income. The association between PFOA and HDL-C was not statistically significant with or without the inclusion of household income, or for the fasting or non-fasting participants. The positive, linear association between PFOS and HDL-C was statistically significant with and without the inclusion of household income, and for both the fasting and non-fasting participants.