Document LKbOJz2jgnyEywX4BymOO50NQ

*^ Int Arch Occup Environ Health DOI 10.1007/s00420-007-0213-0 ORIGINAI \ R I II 1 I P-1 37J $ Assessment of lipid, hepatic, and thyroid parameters with serum perfluorooctanoate (PFOA) concentrations in fluorochemical production workers Geary W. Olsen Larry R. Zobel Received: 4 September 2006/Accepted: 1 June 2007 Springer-Verlag 2007 Abstract Objectives Perfluorooctanoic acid (PFOA) results in peroxisome proliferator mediated effects in rats and mice resulting in hypolipidemia but not in monkeys. Counter intuitive modestly positive associations between PFOA and cholesterol levels in production workers have been incon sistently reported. The purpose of this assessment was to examine this association in male workers who manufac tured or used PFOA at three facilities. Methods Subjects were male employee voluntary partic ipants of a fluorochemical medical surveillance program who provided blood samples for serum measurement of PFOA (perfluorooctanoate) and various lipid, hepatic, and thyroid parameters. Statistical analyses included multiple and logistic regression and analysis of covariance. Results A total of 506 employees, who did not take cholesterol-lowering medications (93% of all male par ticipants), were analyzed. Serum PFOA concentrations ranged from 0.007 to 92.03 pg/ml [arithmetic mean 2.21 pg/ml (95% confidence interval 1.66-2.77), median 1.10 pg/ml]. Adjusted for age, body mass index, and alcohol usage in regression analyses, PFOA was not sta tistically significantly {P > 0.05) associated with total cholesterol or low-density lipoproteins (LDL). High-den sity lipoproteins (HDL) were significantly negatively (P < 0.01) associated with PFOA for the three facilities combined but not by individual sites, indicating the overall result was likely a consequence of residual confounding due to different demographic profiles at these sites. Serum triglycerides were significantly positively associated with G. W. Olsen (E3) - L. R. Zobel Medical Department, 3M Company, Mail Stop 220-6W-08, St Paul, MN 55144, USA e-mail: gwolsen@mmm.com PFOA but not consistently by locations. There were no statistically significant associations observed between PFOA and hepatic enzymes for the three facilities com bined although some modest positive associations were observed between PFOA and hepatic enzymes at one of the three facilities. Analyses of all locations showed no associations with TSH or T4 and PFOA. A negative association was observed for free T4 and positive associ ation for T3; however, the findings were well within these assays' normal reference ranges. Conclusion There was no evidence that employees' ser um PFOA concentrations were associated with total cho lesterol or LDL. A negative association with HDL was explained by demographic differences across the three locations. Several explanations are offered for the incon sistent triglyceride associations with PFOA including both methodological as well as biological possibilities. Keywords Perfluorooctanoate PFOA Fluorochemicals Cholesterol Lipids Introduction The ammonium salt of perfluorooctanoic acid (APFO), which rapidly dissociates in blood to perfluorooctanoate (PFOA, C7F |5COO- ), has been used as a processing aid in the production of various fluoropolymers. The widespread presence of PFOA in the blood of the general population (Calafat et al. 2006) has resulted in assessments of sources of exposure including industrial production, indirect sour ces including atmospheric production, and transport of telomer precursors (De Silva and Maybury 2006). As a manufacturer and user of APFO until its phase-out beginning in 2000, the 3M Company (3M) has reported i ) Springer P-2 Int Arch Occup Environ Health several periodic medical surveillance analyses of its fluorochemical production workers at its Antwerp, Belgium; Cottage Grove, MN; and Decatur, AL manufacturing facilities (Ubel et al. 1980; Gilliland and Mandel 1996; Olsen et al. 1998, 2000, 2003a). These analyses compared workers' clinical chemistry and hormone results in relation to their serum measurements of either PFOA or perfluorooctanesulfonate (PFOS, C8F 17S 0 3). In one of the aforementioned analyses, Olsen et al. (2003a) reported a positive association between PFOA and serum cholesterol and triglycerides among participants of a fluorochemical medical surveillance program conducted in 2000 at the 3M Antwerp and Decatur facilities. They considered this a spurious finding given the toxicological evidence in mice and rats that shows PFOA, a peroxisome proliferator alpha receptor agonist (PPARa), exerts effects including increased beta-oxidation of fatty acids, inhibition of the secretion of very low-density lipoproteins and cho lesterol from the liver, and a reduction of cholesterol and triglycerides in serum and accumulation of lipids in the liver (Maloney and Waxman 1999; Xie et al. 2003; Ken nedy et al. 2004). No associations were reported between PFOA and either serum cholesterol or triglycerides for the Cottage Grove male employees who participated in the same fluorochemical medical surveillance program in 2000 (Olsen et al. 2003c). In response to these occupational findings, Costa (2004) reported preliminary cross-sectional analyses of data col lected from 2000 to 2004 of approximately 40 (per year) Italian fluorochemical production workers. Costa observed a mode&t increase of total cholesterol in workers exposed to PFOA. There was no increase of other lipids, including triglycerides, but the fraction of non-high density lipopro tein (non-HDL) cholesterol appeared elevated. Costa sug gested his findings might be consistent with the hypothesis that PFOA could influence cholesteryl ester transfer protein (CETP), a plasma glycoprotein that facilitates the transfer of cholesteryl esters from apolipoprotein A-containing lipoprotein (HDL) to apolipoprotein B-containing lipo proteins (Brousseau et al. 2004). Kaplan (2004) summarized a cross-sectional analysis of 782 male and 243 female (combined eligible population 1,863) workers with potential exposure to PFOA at the Du Pont de Nemours Co. (DuPont) Parkersburg, West Virginia facility. The highest measured PFOA concentration reported was approximately 10 pg/ml with the average less than 1 pg/ ml. Statistically significant positive associations were iden tified for total cholesterol, low-density lipoproteins (LDL) and triglycerides with serum PFOA concentrations although the percent variation explained was also low. No association, however, was seen with the HDL fraction thereby not sup porting a role between PFOA and CETP. Worker EKG results and C-reactive protein measurements were also not associated with PFOA. Although Kaplan offered no addi tional hypotheses or suggested causal models (Hernn et al. 2002), PFOA has been shown to bind or be carried on blood albumin (Han et al. 2003) and beta-lipoproteins (KerstnerWood et al. 2003) in the rat, monkey and human; thus, a positive non-causal correlation remains a possibility. Re-examination of the Olsen et al. (2003a) analyses suggested some limitations as they would relate to testing the hypothesis that PFOA is positively associated with total cholesterol and its non-HDL sub-fractions. First, the anal yses were not stratified by cholesterol lowering medication status. A positive association could be masked by inclusion of subjects whose serum cholesterol levels have been re duced by medication if such an increase was, in part, associated with higher PFOA concentrations. Second, LDL calculations were not restricted to those instances where serum triglycerides were <400 mg/dl The potential for bias in the LDL calculation steadily increases with higher tri glyceride levels (Nakanishi et al. 2000). Third, the Antwerp and Decatur facility study concentrated primarily on PFOS, not PFOA. Both PFOS and PFOA have been shown to result in hypolipidemia in rats at high concentrations and thus the causal model hypothesized in these earlier analyses con centrated on testing for this effect, not hyperlipidemia. Fi nally, there was no overall analysis of the three manufacturing facilities (Antwerp, Belgium; Cottage Grove, MN; and Decatur, AL) as the Cottage Grove anal ysis has historically been reported separately (Ubel et al. 1980; Gilliland and Mandel 1996; Olsen et al. 2000, 2003a) due to differences in fluorochemical production activities. The purpose of this study was to analyze the 3M fluoro chemical medical surveillance program data collected in 2000 in order to examine the hypothesis that PFOA may be positively associated with increased cholesterol, LDL, and triglyceride levels across the three 3M fluorochemical pro duction facilities. Analyses focused on male employees who self-reported that they were not taking cholesterol lowering medications in order to minimize unexplained bias intro duced by including participants taking such medication. Because PFOS concentrations were not previously associ ated with lowered serum cholesterol (Olsen et al. 2003a), PFOS was not considered an important potential con founding variable in these PFOA analyses. Three covariates were: age, BMI and alcohol. In addition to the lipids assayed, hepatic enzyme and thyroid tests were also analyzed. Serum triglyceride levels were also considered a potential confounder with hepatic enzyme analyses (Clark et al. 2003). Materials and methods The 2000 fluorochemical medical surveillance program at 3M was available on a voluntary basis to all Antwerp, <S Springer 3 P-3 Int Arch Occup Environ Health Decatur, and Cottage Grove chemical plant employees and standard (tetra hydro-PFOS) were added. Samples were those with site-wide responsibilities (e.g., environmental, analyzed by liquid chromatography/tandem mass spec health and safety workers). Informed consent was obtained trometry using a PE Sciex API 3000 (Applied Biosystems, to measure specific fluorochemical concentrations in the Foster City, CA). The instrument was operated in the blood of program participants, including PFOA. The multiple reaction monitoring mode under optimized con number of individuals eligible (i.e., potential to have had ditions for detection of PFOA ions formed by turbionspray occupationally related exposure) for the program included ionization. All PFOA measurements were reported above approximately 340 Antwerp, 400 Decatur, and 200 Cottage the lower limit of quantitation (5.8 ng/ml). Grove employees. . Statistical analyses included analysis of variance, anal Clinical chemistry variables considered in the analyses ysis of covariance, logistic regression, and multiple were: alkaline phosphatase (IU/1), gamma glutamyl trans regression using JMP (Cary,, NC) and Stata (College Sta ferase (GGT, IU/1), aspartate aminotransferase (AST, IU/1), tion, TX) software. Age, BMI and alcohol (drinks per day) alanine aminotransferase (ALT, IU/1), total and direct bili were considered covariates in the multivariable models. rubin (mg/dl), blood glucose (mg/dl), cholesterol (mg/dl), For analysis of hepatic enzymes, serum triglycerides were LDL (mg/dl), HDL (mg/dl), and triglycerides (mg/dl).: also considered a covariate. Distributions were examined to These measurements were.performed at Allina Laboratories assess normality using non-transformed variables and their (St Paul, MN). LDL was an indirect calculation using the transformations (log, square root, inverse). In general, log Friedewald formula (Friedewald et aL 1972) [LDL = total transformations improved normality assumptions of re cholesterol - HDL - (triglycerides/5)] when serum trigly sponse and explanatory variables and thus multiplicative cerides did not exceed 400 rtig/dl. Thyroid stimulating - models (log transformations of explanatory and response hormone (TSH; IU/ml); serum thyroxine:(T4; pg/dl); free variables) were considered applicable. For the log trans thyroxine (free T4; ng/dl) and serum triiodothyronine (T3; formation of alcohol, 0,1 was added to drinks per day to ng/dl) were also analyzed. These thyroid related hormones prevent the log of 0. Goodness of ;fit statistics, multicol- were measured by LabCorp (Kansas City, MO): Thyroid: linearity, and residual plots were examined to detect model hormone analyses excluded''five individuals who were a l , inadequacies. Crude and adjusted odds ratios for PFOA ready diagnosed with thyroid-related conditions and likely categorized by deciles were determined via logistic taking medication. Results, however, did not differ if these regression analyses,for reference range values of the re individuals were'included in the analyses. sponse variable. , PFOA concentrations were also .compared to frequency, A comprehensive report is publicly available elsewhere of the metabolic syndrome in the study participants. Clin that details the present study methods and results (Olsen ical identification of the metabolic syndrome in men in and Zobel 2006). . cludes any three of the following: waist circumference >40 in. (surrogate BMI > 30); triglycerides >150 mg/dl; HDL < 40 mg/dl; blood pressure > 130/85 mmHg; aiid Results fasting glucose > 110 mg/dl (Expert Panel 2001). The present study definition of the metabolic syndrome in A total of 552 employees (Antwerp = 206; Cottage cluded any three of the following four: BMI > 30; trigly Grove =131; Decatur = 215) participated from these cerides > 150 mg/dl; HDL < 40 mg/dl; and fasting facilities representing approximately 60% of Antwerp, glucose > 110 mg/dl. ' 1 50% of Decatur and 65% of Cottage Grove eligible At the time of the analyses that were done in 2000-2001 employees. Demographic characteristics of those eligible by Tandem Labs (formerly Northwest Bioanalytical, Salt employees who did not participate are not known although Lake City, UT), the analytical methods used were com substantial differences between participants and non-par parable to those reported by Hansen et al. (2001). The ticipants are unlikely given the routine practice of offering analytical method consisted of a liquidiliquid extraction voluntary medical surveillance programs at these facilities procedure followed by evaporation and reconstitution of for more than 20 years. the extract residue with 30:70 20 mM ammonium acetate Of the 552 employee participants at these 3 facilities, in water:20 mM ammonium acetate in methanol (v/v). 506 (92%) self-reported that they did not take cholesterol Eight or more calibration standards were prepared on the lowering medications: 196 (95%) for Antwerp, 122 (93%) day of each run by adding 100 pi of blank human serum forCottage Grove, and 188 (87%) for Decatur. Results will and 400 pi of 50 mM ammonium acetate in water to focus on these 506 participants. polypropylene tubes. After a brief vortex mixing, 10.0 pi Presented in Table 1 are measures of central tendency of the appropriate spiking solution was added. In addition, for the 506 participants for PFOA, PFOS, demographic blank serum samples, both with and without internal factors and the clinical chemistries. The number and per- ) Springer Int Arch Occup Environ Health X) oo Springer Table 1 Mean, standard deviation, median and range of PFOA, PFOS, demographic factors and clinical chemistry results, by location Total (N = 506) Antwerp (N = 196) Cottage Grove (N = 122) Mean SD Median Range Mean SD Median Range Mean SD Median Range PFOA PFOS Age BM1 Alcohol Glucose Cholesterol LDL HDL Triglycerides Aik Phos AST ALT AST/ALT GGT Total Bil Direct Bil TSH T4 Free T4 T3 2.21 1.05 40 27.4 0.6 91 214 136 49 159 66 25 30 0.98 28 0.9 0.1 2.4 8.2 1.1 127 6.40 1.10 0.97 0.72 9 39 5 26.7 0.9 0.3 19 91 41 211 36 133 13 46 113 128 18 64 7 24 15 26 0.12 0.97 22 22 0.3 0.8 0.06 0.1 3.4 1.9 1.4 8.2 0.2 1.1 23 125 0.01-92.03 0.02-6.24 21-67 17.2-52.1 0.0-6.4 31-259 105-331 37-235 18-121 24-796 21-160 10-69 6-103 0.70-1.82 6-314 0.3-2.3 0.0-0.7 0.03-65.3 4.2-12.0 0.6-1.8 78-300 1.02b 0.95" 37b, c 24 7b, c l . l b' c 84b- c 217 139b 55b' c 120b' c 60b' c 23b. c 23b' c 1.04b' c 23b' c ! ,0b' 0.1" 2.0C 8.2 l.lc 13 1b- c 1.06 0.97 8 3.0 1.1 17 41 37 15 83 14 6 10 0.12 17 0.3 0.05 1.6 1.4 0.2 19 " Statistically significantly (P < 0.05) different than Antwerp b Statistically significantly (P < 0.05) different than Cottage Grove c Statistically significantly (P < 0.05) different than Decatur 0.65 0.55 36 24.5 0.9 87 216 134 53 100 61 22 21 1.02 18 1.0 0,1 1.7 8.2 1.1 130 - 0.01-7.04 0.04-6.24 21-58 17.5-34.7 0.0-6.4 31-131 105-331 43-235 26-121 34-731 21-113 13-58 8-71 0.81-1.47 6-111 0.4-2.3 0.0-0.4 0.03-19.4 4.2-12.0 0.6-1.6 87-185 4.63"- c 0.86c. .41" 29.9"' c 0.7"' c 100"' c 210 130" 46" 187" 65"' c 25" 34" 0.94" . 31a 0.9"' c 0.1 2.4 7.9C 1.1 125" 12.53 0.98 9 4.8 0.7 23 39 32 11 139 15 8 17 0.09 32 0.3 0.02 1.4 1.1 0.1 30 0.95 0.45 40 29.5 0.5 95 206 125 45 142 63 24 29 0.92 22 0.8 0.1 2.0 7.9 1.1 121 0.01-92.03 0.03-4.79 24-67 19.7-52.1 0.0-3.0 59-259 130-311 37-208 18-77 24-725 28-107 10-54 13-95 0.70-1.25 7-314 0.4--2.3 0.0-0.2 0.03-9.4 4.9-11.6 0.8-1.8 78-300 Decatur (N = 188) Mean SD Median 1.89b 1.61 1.51 1.29"' b 0.92 1.00 42" 9 43 28.6"' b 4.4 27.5 0.1"' b 0.3 0.0 14 91 214 41 211 136 36 133 44" 10 42 182" 110 164 73"'b 20 69 26" 8 25 34" 16 30 0.95" 0.11 0.93 30" 17 27 0.7"' b 0.2 0.7 0.1" 0.07 0.1 2.8" 5.2 1.9 1.4 8.5 1.1" 0.2 1.1 125" 22 120 Range 0.04-12.70 0.06-4.17 26-63 17.2-50.1 0.0-2.0 74-184 121-319 47-225 24-82 32-796 26-160 13-69 6-103 0.75-1.82 7-144 0.3-1.5 0.0-0.7 0.03-65.3 4.6-11.4 0.6-1.5 86-196 P-5 Int Arch Occup Environ Health cent of employees by specific reference points for demo graphic factors and clinical chemistry findings are pre sented in Table 2. Besides their younger age, there were substantially fewer Antwerp employees (5%) who would be categorized as obese (BMI > 30) compared to Cottage Grove (44%) or Decatur (32%) employees. More than 40% of Antwerp and Cottage Grove employees reported drink ing one alcoholic beverage per day or more compared to only 1% of Decatur employees. There were regional cultural/lifestyle practices that likely account for this differ ence in alcohol consumption. Other statistically significant differences between the three locations were observed for blood glucose, HDL, triglycerides, alkaline phosphatase, ALT, total bilirubin, and the `metabolic syndrome'. The higher percentage of Antwerp male employees with total bilirubin >1.5 mg/dl has been attributed to the likely greater prevalence of Gilbert's syndrome (Olsen et al. 1999). Only 1% of the Antwerp employees were catego rized as having the `metabolic syndrome' compared to 20 and 13% among the Cottage Grove and Decatur employ ees, respectively. Serum PFOA concentrations for the 506 employees ranged from 0.007 to 92.03 pg/ml [arithmetic mean 2.21 pg/ml (95% confidence interval 1.66-2.77), median 1.10 pg/mlj. PFOA results are categorized by deciles in Table 3. Mean PFOA decile concentrations ranged from the lowest decile of 0.06 pg/ml (range 0.007-0.13 pg/ml) to the highest decile of 12.15 pg/ml (range 3.71-92.03 pg/ ml) with corresponding median values of 0.06 and 4.94 pg/ ml, respectively. The distributions of demographic factors by PFOA decile are presented in Table 4. There were higher per centages of Antwerp employees in the first three deciles and lower percentages in the highest two deciles (9 and 10). Likewise, there were lower percentages of Decatur employees in the first three deciles and higher percentages in the upper deciles (6 10), in particular decile 9. Because of these disparities, mean BMI values were greater in the Table 2 Number of employees (%) by location for reference points of demographic factors and clinical chemistry results Percentage values are given in parentheses 3 Chi square test of significance b See text for description Age < 40 years . BMI > 3 0 Alcohol > 1 drink/day Glucose > 110 mg/dl Cholesterol > 200 mg/dl Cholesterol > 240 mg/dl HDL < 40 mg/dl LDL > 130 mg/dl Triglycerides > 150 mg/dl Alkaline phosphatase > 120 IU/1 AST > 40 IU/1 ' ALT > 40 IU/1 GGT > 40 IU/1 Total bilirubin > 1.5 mg/dl Direct bilirubin > 0.4 mg/dl TSH <0.35 itlU/ml >5.5 /rIU/ml T4 <4.5 pg/dl >12.0 pg/dl Free T4 <0.70 ng/dl >1.53 ng/dl T3 <60 ng/dl >181 ng/dl Metabolic syndrome6 Antwerp . (N = 196) 127 (65) 9(5) 92 (47) 10(5) 119 (61) 60(31) 16(8) 106 (55) 46 (23) 0(0) 4(2) 14(7) 25 (13) 13 (7) 0(0) 1 G) 5 (3) 1 (1) 0(0) 1 G) 3 (2) 0(0) 2(1) 2(1) Cottage Grove (N = 122) 57 (47) 54 (44) 49 (40) 22 (18) 69 (57) 32 (26) 36 (30) 53 (48) 58 (48) 0(0) 8 (7) 34 (28) 27 (22) 1 (1) 0(0) Decatur ( N = 188) 76 (40) . . 61 (32) 2(1) 16(9) 114 (61) 50 (27) 72 (38) 100 (55) 103 (55) 5 (3) 11 (6) 51 (27) 34 (18) 0(0) 2(1) 1 G) 5 (4) 1 G) 8 (4) 0(0) 0(0) 0(0) 0(0) 0(0) 1 G) 0(0) 3 (3) 24 (20) 2(1) 1 G) 0(0) 2(1) 25 (13) P value3 0.0001 0.0001 0.0001 0.001 0.72 0.60 0.0001 0.40 0.0001 0.01 0.09 0.0001 0.09 0.0001 0.18 0.92 0.60 0.46 1.0 0.49 0.60 1.0 0.46 0.0001 Springer P-6 Int Arch Occup Environ Health Table 3 Number of employees, arithmetic mean, 95% confidence interval (95% Cl), median and range, by PFOA decile PFOA Decile N PFOA Mean 95% Cl Median Range 1 51 0.061H 0.05-0.07 0.06 0.007-0.13 2 51 o ^o 1- j 0.19-0.21 0.19 0.13-0.29 3 51 0.36"' j 0.35-0.37 0.36 0.30-0.44 4 50 0.55s' j 0.53-0.57 0.54 0.44-0.71 5 51 0.9 lj 0.87-0.94 0.91 0.72-1.10 6 49 1.26* 1.23-1.28 1.25 1.11-1.40 7 50 1.64* 1.60-1.67 1.63 1.42-1.85 8 54 2.17"' > 2.12-2.23 2.18 1.86-2.50 9 49 3.00a_d' j 2.90-3.10 2.96 2.51-3.69 10 50 12.15a_i 7.21-17.10 4.94 3.71-92.03 a-j Statistically significantly (P < 0.05) different than PFOA decile(s) 1, 2, 3,..., 10, respectively upper deciles and alcohol consumption was lower, reflecting the demographic differences seen across the three facilities in Tables 1 and 2. Presented in Figs. 1 and 2 are scatterplots of the natural logs of the blood lipids and PFOA. Pearson correlation coefficients and P values (in parentheses) between PFOA and the lipid parameters were: cholesterol (r = 0.05, P = 0.32), LDL (r = 0.006, P = 0.90), HDL (r = -0.17, P < 0.0001), and triglycerides (r = 0.21, P < 0.0001). Unadjusted and adjusted coefficients for PFOA when re gressed with total cholesterol, LDL, HDL, and triglycerides are presented in Table 5. Analyses are also presented for each location in Table 5. PFOA was not a statistically significant coefficient in the regression models for total cholesterol or LDL. In the regression model for HDL that contained all three locations, the adjusted PFOA coefficient was statistically significant (P - 0.01) but only explained 1% of the variance in the model. Separate models analyzed for each of the three locations showed no statistically sig nificant findings between PFOA and HDL. The model for triglycerides for the combined three locations indicated a statistically significant positive coefficient (P < 0.0001) for PFOA which explained approximately 4% of the variance of the response variable. Stratified by location showed Antwerp with a statistically significant positive PFOA coefficient (P < 0.0004) and Decatur had a marginally positive coefficient for PFOA (P = 0.07). No statistically significant coefficient for PFOA (P = 0.38) was found with triglycerides for the Cottage Grove location. Presented in Table 6 are the mean and 95% confidence intervals (Cl) values for cholesterol, LDL, HDL, and tri glycerides by PFOA deciles, adjusted for age, BMI, and alcohol. Mean serum triglyceride levels were highest in the upper three PFOA deciles. Table 7 presents adjusted odds ratios and 95% confi dence intervals by reference range cutoff points listed in Table 2, using the lowest PFOA decile as the reference. Serum PFOA concentrations were not associated, posi tively or negatively, with cholesterol or LDL. For HDL < 40 mg/dl, the non-adjusted odds ratio for the highest decile was 3.0 (95% Cl 1.2-7.5). Adjusted for age, BMI and alcohol, the highest decile odds ratio became 2.6 (95% Cl 1.0-6.8) (Table 5). Adjusted also for location, this odds ratio became 1.8 (95% Cl 0.7-4.8). Adjusted odds ratios for triglyceride levels >150 mg/dl were also highest Table 4 Distribution of demographic factors by PFOA decile PFOA Antwerp Cottage Grove Decatur Age BMI Decile N{%) N (%) N(%) Mean (SD) Mean (SD) BMI > 30 N (%) Drinks/day Mean (SD) 1 28 (55) 17 (33) 6(12) 39 (11) 27.1 (4.8) 13 (25) 0.7 (0.7) 2 25 (49) 13 (26) 13 (25) 41 (9) 26.7 (4.1)' 7 (14) 0.7 (0.9) 3 28 (55) 11 (22) 12 (23) 38 (8) 26.4 (3.9)1' J 8(16) 0.7 (1.0) 4 20 (40) 13 (26) 17 (34) 38 (10) 27.8 (4.3) 13 (26) 0.7 (1.0) 5 22 (43) 11 (22) 18 (35) 40 (10) 27.2 (5.0) 8(17) 0.7 (10) 6 21 (43) 7 (14) 21 (43) 40 (9) 27.4 (4.8) 12 (24) 0.7 (1.0) 7 20 (40) 9 (18) 21 (42) 39 (9) 26.9 (3.9)' 10 (20) 0.7 (0.9) 8 18 (33) 11 (20) 9 9(18) 7 (14) 25 (46) 33 (67) 41 39 (10) 27.5 (5.4) 17 (31) 0.6 (0.9) (9) 28.9 (4 9)". c, s 21 (43) 0.3 (0.5)a~h 10 5 (10) 23 (46) 22 (44) 39 (9) 28.3 (4.0)c 15 (30) 0.5 (0.7) P < 0.0001k P < 0.031 Statistically significantly (P < 0.05) different than PFOA decile 1, 2, 3,..., 10, respectively k- 'm P value associated with Chi square test for location, BMI distribution and >1 drink/day, respectively >1 drink/day N (%) 21 (41) 17 (33) 15 (29) 13 (26) 16 (32) 14 (29) 14 (28) 16 (30) 5 (10) 12 (24) P < 0.14"1 Springer 1 V* Int Arch Occup Environ Health p .7 - 6.0 -4.0 -2.0 0.0 2.0 4.0 6.0 in PFOA Fig. 1 Scatterplots of the natural log PFQA (ng/ml) by the natural log total cholesterol and LDE .. Fig, 2 Scatterplots of the natural log PFOA (ng/ml) by the natural log HDL and triglycerides , for PFOA deciles 8-10 as seen with adjusted .mean tri glyceride values reported in Table 7. Presented in Figs. 3 and 4 are scatterplots of natural logs of hepatic clinical chemistries by PFOA. Pearson correla tion coefficients and P values (in parentheses) between PFOA and the hepatic parameters were: alkaline phos phatase (r = 0.08, P = 0.06), AST (r = -0.01 P = 0.83), ALT (r = 0.005, P < 0.005), and GGT (r = 0.02, P < 0.02). Analyses presented in Table 8 of the regression models showed marginal statistically significant coeffi cients for PFOA when adjusted for age, BMI, and alcohol for ALT (P = 0.06) and GGT (P = 0.05) but not when, adjusted for age, triglycerides, and alcohol for ALT (P = 0.40) and GGT (P --0.55). Total bilirubin was sta tistically significantly negatively associated with PFOA regardless of covariates used. Stratified by location, Decatur had marginally statistically significant positive coefficients, regardless of covariates adjusted, ranging be tween 0.01 < P < 0.07 for alkaline phosphatase, ALT, GGT, and total bilirubin. The amount of variance of the hepatic response variables explained by PFOA in these models was minimal ranging from <1 to 3%. No statisti cally significant PFOA coefficients for the hepatic enzymes were observed in either the Antwerp or Cottage Grove regression models. No discernable trends in adjusted mean hepatic enzyme values were apparent when analyzed by deciles (Table 9). There were no statistically significant odds ratios (non adjusted or adjusted) for the reference points AL T > 40 IU/1 or GGT > 40 IU/1 when compared to decile 1 (Table 10). Odds ratios for alkaline phosphatase, AST, and total bilirubin are not shown because of the failure of the logistic models to converge because of the very few data points that were out-of-reference range (Table 2) for these clinical parameters. Presented in Figs. 5 and 6 are scatterplots of natural logs of thyroid hormones by PFOA. Pearson correlation coef ficients and P values (in parentheses) between PFOA and the thyroid hormones were: TSH (r = 0.08, P = 0.07), T4 (r = -0.04, P = 0.32), free T4 (r = -0.14, P < 0.002), and T3 (r = 0.07, P = 0.11). For all locations combined, there were no statistically significant adjusted coefficients for PFOA in the models except for Free T4 (negative coeffi cient for PFOA) and T3 (positive coefficient for PFOA) (Table 11). However, the full models only explained 5 and 2% of the variance of free T4 and T3, respectively. The thyroid results were considered not clinically relevant as results from these models were well within normal refer ence ranges for the four thyroid parameters when serum PFOA concentrations were predicted in a model to range <) Springer p .8 Int Arch Occup Environ HeaLn - Table 5 Non-adjusted and adjusted natural log (In) PFOA coefficients with In lipid measurements :1 See study-methods. Adjusted for Ln age, Ln BMI, Ln alcohol Ln cholesterol All locations Antwerp Cottage Grove Decatur Ln LDL All locations Antwerp Cottage Grove Decatur Ln HDL All locations Antwerp Cottage Grove Decatur Ln triglycerides All locations Antwerp Cottage Grove Decatur Ln PFOA Non-adjusted Coefficient SF. 0.0059 0.0051 0.0034 0.0221 0.0012 -0.0037 -0.0022 0.0258 -0.0307 -0.0057 -0.0153 -0.0256 0.0892 0.0840 0.0343 0.0715 0.0060 0.0106 0.0089 0.0139 0.0089 0.0157 0.0139 0.0199 0.0079 0.0136 0.0122 0.0149 0.0185 0.0288 0.0316 0.0400 P value 0.32 0.63 0.70 0.11 0.89 0.81 0.87 0.20 0.0001 0.68 0.21 0.09 0.0001 0.004 0.28 0.08 Ln PFOA Adjusted" Coefficient SE 0.0076 0.0130 0.0021 0.0266 0.0059 0.0096 0.0100 0.0141 0.0021 0.0106 0.0049 0.0302 0.0090 0.0147 0.0145 0.0200 -0.0183 -0.0095 -0.0192 -0.0207 .0069 0.0131 0.0120 . 0.0141 0.0711 0.0169 0.0980 0.0270 0.0280 0.0314 0.0689 . 0.0376 P value 0.20 0.18 0.83 0.06 0.81 0.47 0.73 0.13 0.01 0.47 0.11 0.14 0.0001 0.0004 0.38 0.07 between 0.005 and 100 pg/ml (Table 12). Presented in Table 13 are the mean thyroid hormone-related values adjusted for age, BMI and alcohol. The mean TSH value in the fourth decile of Table 13 is influenced by one subject whose TSH value was 65.3 /tIU/ml (see overall results in Table 1) who was not diagnosed at the time as hypothy roid. If removed, the mean of the fourth decile became 2.15 /rlU/ml and the decile statistically significant differ ences with this fourth decile in Table 13 disappear. The adjusted mean for the highest decile for free T4 was sta tistically significantly lower than that of the first decile. There were no statistically significant differences between decile-adjusted means for T3 in Table 13. Because so few thyroid values were out-of-reference range (Table 2), the findings from the logistic analyses are not presented be cause of the models' lack of convergence. PFOA was not associated with the metabolic syndrome. Age-adjusted odds ratios (95% Cl in parentheses) for the Table 6 Adjusted mean and 95% confidence intervals (95% Cl) for lipid clinical chemistry results, by PFOA decile PFOA Cholesterol LDL HDL Triglycerides Decile Mean 95% Cl Mean 95% Cl Mean 95% Cl Mean 1 214 203-225 137 127-147 50* 46-53 2 211 199-222 135 125-145 5F 48-54 3 209 198-220 128 118-138 5F 48-54 4 210 199-222 133 123-143 50* 46-53 5 217 206-228 140 130-150 48 . 45-51 6 218 206-229 141 130-151 48 45-51 7 214 203-225 133 123-143 501 47-53 8 215 204-226 136 126-146 47 44-50 9 221 210-233 140 130-150 48 44-52 10 216 204-227 133 123-144 44a_d 41-47 Adjusted for age, BMI and alcohol Statistically significantly (P < 0.05) different than PFOA decile(s) 1, 2, 3,..., 10, respectively 145* 124h~j 153* !4? 162j ' 160* 158J 172 165* 208a_g' 1 95% Cl 116-173 95-153 124-182 116-175 133-191 131-190 128-187 144-201 135-194 179-238 Springer In GGT T P-9 Int Arch Occup Environ Health Table 7 Adjusted odds ratios (OR) and 95% confidence intervals (95% Cl) for lipid clinical chemistry reference points, by PFOA decile PFOA Choi > 200 mg/dl LDL > 130 mg/dl HDL < 40 mg/dl Triglycerides > 150 mg/dl Decile ORa 95% Cl ORb 95% Cl ORa 95% Cl ORb 95% Cl ORa 95% Cl ORb 95% Cl ORa 95% Cl ORb 95% Cl 1 1.0 _ 1.0 - 1.0 - 1.0 2 0.4 0.2-1.0 0.4 0.2-1.0 0.7 0.3-1.6 0.7 3 0.9 0.4--2.0 0.9 0.4--2.0 0.7 0.3-1.6 0.7 4 0.9 0.4-2.1 0.9 0.4-2.0 0.8 0.4--1.8 0.8 5 1.7 0.7^1.0 1.6 0.7-3.9 1.4 0.6-3.1 0.4 6 1.0 0.4-2.2 0.9 0.4-2.1 0.9 0.4--2.0 0.9 7 0.8 0.4-1.9 0.8 0.3-1.8 0.7 0.3-1.5 0.7 8 1.0 0.4-2.2 0.9 0.4-2.1 1.1 0.5-2.4 1.1 9 1.4 0.6-3.3 . 1.3 0.6-3.1 1.2 0.5-2.7 1.2 10 1.1 0.5-2.6 1.1 0.5-2.6 1.2 0.5-2.8 1.4 a Adjusted for age, BMI and alcohol b Adjusted for age, BMI, alcohol and location - 0.3-1.7 0.3-1.6 0.4-1.9 0.6-3.2 0.4-2.1 0.4-1.6 0.5-2.5 0.5-2.8 0.6-3.3 1.0 1.1 0.5 2.0 1.0 1.7 0.6 1.4 0.9 2.6 - 0.4-3.2 0.1-1.5 0.8-5.4 0.4-2.9 0.7^t,7 0.2-1.8 0.5-3.7 0.3-2.4 1.0-6.8 1.0 1.0 0.4 1.7 0.8 1.4 0.5 1.0 0.6 1.8 - 0.4-3.0 0.6-4.7 0.3-2.4 0.5-3.9 0.1-1.4 0.4-2.8 0.2-1.7 0.7-4.8 1.0 0.7 1.0 1.3 1.2 1.7 0.9 2.7 2.4 2.4 - 0.3-1.8 0.4-2.4 0.5-3.1 0.5-3.0 0.7^1.0 0.4-2.2 1.2-6.5 1.0-5.9 1.0-5.8 1.0 0.6 0.9 1.1 1.0 1.3 0.7 2.1 1.7 1.8 - 0.2-1.6 0.4-2.2 0.4-2.6 0.4-2.5 0.5-3.2 0.3-1.8 0.9-5.2 0.7--4.3 0.8^1.4 6th, 7th, 8th, 9th and 10th PFOA deciles were 1.0 (0.3 3.5), 0.5 (0.1-2.0), 0.9 (0.3-3.0), 1.1 (0.3-3.6) and 1.0 (0.3-3.6), respectively. Other analyses included those subjects (n = 46) who self-reported cholesterol lowering medication usage. These 46 subjects, compared to the 506 non-prescribed subjects,' had comparable mean PFOA concentrations (1.98 pg/ml vs. 2.21 pg/ml) but were statistically significantly older (49 vs. 40) and had higher mean BMI (28.8 vs. 27.4), serum glucose (103 vs. 91), triglycerides (226 vs. 159), and sev eral liver enzyme results (alkaline phosphatase 73 vs. 66, ALT 36 vs. 30, GGT 36 vs: 28). Cholesterol (221 vs. 214), LDL (134 vs. 136), and HDL (47 vs. 49) were not statis tically significantly different between the two groups. No ---------------------------------------------- A S * * . A W **' r * * * 2.0 C0 * *' :. r t- s m * X . msn 1 * * f j r . m. * v* * * -------------,,-------------,,------------- .,-------------i1------------1--r-&frr9--1------------ 1.----------- 11----------- 1.-----11-----.!----- - 6.0 - 4.0 - 2.0 0.0 2.0 4.0 6.0 In PFOA Fig. 3 Scatterplots of the natural log PFOA (ng/ml) by the natural log alkaline phosphatase and AST ' 1 ' 1--------1--------1-------- .-------- 1-------- ! 0.0 -6-0 *40 -2.0 0.0 ' In PFOA 1----- '-----'-----'----- 2.0 4.0 6.0 Fig. 4 Scatterplots of the natural log PFOA (ng/ml) by the natural log ALT and GGT Springer / p. 10 Int Arch Occup Environ Health Table 8 Non-adjusted and adjusted natural log (In) PFOA coefficients with In hepatic clinical chemistry measurements Non-adjusted Ln PFOA Coefficient Ln alkaline phosphatase All locations 0.0155 SE 0.0082 Antwerp -0.0025 0.0137 Cottage Grove -0.0141 0.0113 Decatur 0.0394 0.0191 P value 0.06 0.85 0.21 0.04 Ln AST All locations Antwerp Cottage Grove -0.0018 -0.0048 -0.0281 0.0086 0.0137 0.0141 0.83 0.73 0.05 Decatur 0.0205 0.0200 0.31 Ln ALT All locations Antwerp Cottage Grove Decatur 0.0402 -0.0122 -0.0131 0.0954 0.0143 0.0220 0.0215 0.0300 0.005 0.58 0.54 0.002 Ln GGT All locations Antwerp Cottage Grove Decatur 0.0409 0.0174 0.0170 0.0307 -0.0088 0.0292 0.0754 . 0.0344 0.02 0.58 0.76 0.03 Ln total bilirubin All locations -0.0406 Antwerp -0.0117 3 Study methods. Adjusted for Ln age, Ln BMI, Ln alcohol b Study methods. Adjusted for Ln age, Ln triglycerides, Ln alcohol Cottage Grove Decatur -0.0060 -0.0528 0.0101 0.0178 0.0138 0.0203 0.0001 0.51 0.66 0.01 Adjusted3, b Ln PFOA Coefficient SE P value 0.00933 0.0037b -0.0060 -0.0170 -0.0127 -0.0140 0.0460 0.0394 -0.0051 -0.0089 -0.0029 -0.0066 -0.0258 -0.0271 0.0114 0.0062 0.0249 0.0115 -0.0085 -0,0293 -0.0096 , -0.0008 0.0704 0.0581 0.0326 0.0097 0.0269 -0.0047 -0.0198 -0.0233 0.0800 0.0599 -0.0325 -0.0267 -0.0122 -0.0093 -0.0098 -0.0067 -0.0537 -0.0462 0.0081 0.0081 0.0139 0.0140 0.0117 0.0117 0.0192 0.0192 0.25 0.65 0.67 0.22 0.28 0.24 0.02 0.04 0.0086 0.0087 0.0138 0.0142 0.0146 0.0145 0.0203 0.0203 0.55 0.31 0.83 0.64 0.08 0.07 0.57 0.76 0.0132 0.0136 0.0222 0.0222 0.0209 0.0208 0.0287 0.0287 0.06 0.40 0.70 0.19 0.65 0.69 0.02 0.04 0.0166 0.0163 0.0294 0.0295 0.0286 0.0270 0.0344 0.0329 0.05 0.55 0.36 0.87 0.49 0.39 0.02 0.07 0.0099 0.0101 0.0182 0.0188 0.0142 0.0141 0.0209 0.0206 0.001 0.01 0.50 0.62 0.49 0.64 0.01 0.03 <0 Springer Int Arch Occup Environ Health Table 9 Adjusted mean and 95% confidence intervals (95% Cl) for hepatic clinical chemistry results, by PFOA decile Total Direct PFOA Aik Phos AST ALT GGT Bilirubin Bilirubin Decile Mean 95% Cl Mean 95% Cl Mean 95% Cl Mean 95% Cl Mean 95% CI Mean 95% CI 1 66 61-71 26c,f 24-28 29 25-33 32f 27-38 0.9' 2 59sJJ 54-64 25 23-27 29 25-33 25 19-31 0.9' 3 64g 59-69 26e,f 24-28 30 26-34 27 21-33 1.0H 4 65 61-70 24 22-26 28iJ 24-32 28 22-34 0.9' 5 64e 60-69 22a-c'6-i 20-25 27,J 23-31 24' 18-30 0.9 6 66 61-71 22a.c.g-i 20-24 25E,'J 21-28 21" ' 15-27 0.9e 7 -^2b,c,e,h 67-77 26e,f 24-28 31f 27-35 28 22-34 0.9e 8 65s 60-70 26e'f 24-28 29 26-33 29 23-35 0.9e 9 70b 66-75 26e,f 24-28 34d~f 30-38 33a.f 27-39 0.8a_d 10 68b 63-72 24 22-26 34d-r 30-38 30 24-36 0.8e Adjusted for age, BMI, and alcohol Statistically significantly (P < 0.05) different than PFOA decile(s) 1, 2, 3,..., 10, respectively 0.86-1.02 0.84-1.00 0.89-1.06 0.82-0.99 0.79-0.95 0.78-0.95 0.77-0.94 0.77-0.93 0.69-0.85 0.75-0.92 0.1 0.1 0.1 0.1 0.1' 0.1 0.1 0.P 0.1 c'h 0.1 0.09-0.12 0.09-0.12 0.09-0.12 0.08-0.11 0.10-0.13 0.08-0.11 0.08-0.11 0.10-0.13 0.07-0.10 0.08-0.12 significant differences were observed with thyroid hor mones. Twenty percent of these 46 subjects were catego rized as having the metabolic syndrome compared to 7% of those not prescribed cholesterol-lowering medication. Not unexpectedly based on the results already presented, a greater percentage of those prescribed cholesterol-lowering medications were Cottage Grove and Decatur employees (79%) than those not prescribed medications (61%). Analyses with the clinical chemistries were similar whether they excluded or included these 46 employees who self reported cholesterol lowering medications (data not shown). Discussion Based on an analysis of 506 male participants of the 2000 fluorochemical medical surveillance program offered at the 3M Antwerp, Cottage Grove, and Decatur fluorochemical production facilities who self-reported that they did not take cholesterol lowering medications, there was no evi dence that these employees' serum PFOA concentrations were positively, or negatively, associated with serum total cholesterol or LDL. These data analyses support the argument that the PFOA and cholesterol association ob served in an analysis by Olsen et al. (2003a) of Antwerp and Decatur workers in this database could have been a spurious finding or may have been the result of residual confounding between the different demographic popula tions as seen in Tables 1 and 2. Addition of the Cottage Grove workforce, that was actively engaged in the manu facture of the ammonium salt of perfluorooctanoic acid and had the highest serum PFOA concentrations of the three facilities, only further demonstrated the lack of an associ ation between PFOA and cholesterol in this workforce. Unlike rats and mice, there was no reduction in serum cholesterol in cynomolgus monkeys dosed with PFOA (ammonium salt) for 6 months (Butenhoff et al. 2002). A weakly negative association was observed with HDL that was possibly due to uncontrolled (i.e., residual) con founding, based on lower HDL values observed among the Cottage Grove and Decatur workers than the Antwerp workers and their markedly different demographic factors (e.g., BMI). When the analyses were stratified by location, no statistically significant associations were observed be tween HDL and PFOA. In another occupational study, Kaplan (2004) did not report an association between HDL and serum PFOA concentrations. Neither did Emmett et al. (2006a) in a community-based exposure study whose median PFOA concentrations (approximately 0.354 pg/ml) were 60 times higher than that of general population studies (Calafat et al. 2006; Olsen et al. 2003b). To further clarify any possible association with HDL, the A apolipoproteins, which form the major proteins found in HDL, could be measured although this was not part of the present study. Apolipoprotein Al was not associated with serum PFOA of comparable concentrations in a small analysis of Italian production workers (Costa 2004). Serum triglyceride levels were positively associated with PFOA. Although a biological association cannot be ruled out, it is also possible that this association might have been due, at least partially, to residual confounding as a consequence of the disproportionate number of Cottage Grove and Decatur employees with higher PFOA concen trations than the Antwerp employees. The same positive association was also observed between PFOS and serum Springer Int Arch Occup Environ Health . Decile OR" 95% Cl ORb 95% Cl ORc 95% Cl ORd 95% Cl OR' 95% Cl OR" 95% Cl ORb 95% Cl ORc 95% Cl ORd 95% Cl OR' 95% Cl ______________________________________ GGT 5 40 IU/1 Table 10 Adjusted odds ratios (OR) and 95% confidence intervals (95% Cl) for ALT and GGT reference points, by PFOA decile________________________________________________ <N CN r- VO CN Os CN CN <N 7cn cn CN -l mi 4 7cn 7m 7cn 1 d d d d d d d d p r- r- Os p m -3- p d d d d d -- cn cn CN on S 7<N1 CCNN1 7cni 7cn1 7 cn 4 Ii cn cinnii cn1 in 1 d d d d d d d d -fee- * 4.0 p VO 00 ON oo n--d d d d d VO Tf cn d --^ cn CN p in cn VO CN CN1 7CN ccnn1 7 cn 44 3 cn 4 icn 1d d O d d d d d d -4----- In PFOA -6,0-,............-..... ......... p r- r- p 00 d d --d VO VO CN cn d -H --, os Tf oo >q in o >n m 7(N 7 37m 7 7CN VO tVO 1d d d d d d d d d p VO 00 p oo in CN r- vq dd d d --< Os CN Os in CN p -- 7CN 7CN 3 7cn 7CN cn 4 7CN 7Vi vi 1d d d d d d d d d p vO r- p ON in CN r- in vq dd dd d ^t cn ON Os in in oo cn 4 7CN 7cn i ?o 3 3 3 7CN 1d d d d d o o d p oo r- CN CN CN CN ^ VO dddd -- d -- VO ON cn oo ? tin v1o 3r- Io o o o o o o o o r - (N cn cn cn| n oo o d ^ ^ -- o O -N" Os Tf \c \o m 4tn r;i N CN cn 1 1 1 1^ m cn rt- cn in m r- cn dddoooooo os cn cn cn cn d d d *-* -- I CN in riI riI t cn dd Tt (NI cn d -- -I1 ^ d ?~ o` m1 d vto r3*- m7 odo oo oo cn cn m in d dd d ^ ~ o ---- -oC o o "O "O ^j* r~- so oo (eSnl pmii ncnI :I cn cn in cn in 4 Os 4 I d d d ` o d d d d osoooscncnm''t;cN^t; --ddddd--^<N -- <N cn ^ m vo t-- 2235 M is S OJ lU O 4J bxj b b M a 3 "O g33 3 < < <O tT T? F? ^ Z< __________,-----------------,----------- 40M-----------------,-----------------,-----------------; - 6.0 - 4.0 -2.0 0.0 2.0 4.0 6.0 In PFOA Fig. 5 Scatterplots of the natural log PFOA (ng/ml) by the natural log TSH and T3 triglycerides (Olsen et al. 2003c). This prior association with PFOS is inconsistent with the well-established hypolipidemia reported in PFOS-related toxicological studies with concentrations much higher than in the present study (Seacat et al 2002, 2003). On the other hand, PFOA was statistically significantly positively associated with trigly cerides in the high-dose (30/20 mg/kg) monkey group whose steady state serum PFOA concentration was 158 10 pg/ml (range 20^167 pg/ml) (Butenhoff et al. 2002). This association was observed in measurements taken after 1 month of dosing at which time the group mean triglyceride level was significantly higher than con trol values as well as within group pretreatment values. At the end of the study the mean triglyceride was elevated compared to time related controls but not to the animals' pre-treatment values. However, only two primates were evaluated in the high-dose group at end of study. Inspection of individual values for PFOA serum concentration and serum triglyceride values did not reveal any meaningful associations between these two parameters. Of the four lipid measurements assessed in this study, serum triglycerides have three times more intra-individual biological variation than either total cholesterol, LDL, or HDL (Stein and Myers 1994). Serum triglycerides are influenced by obesity, alcohol intake, and inattention to PFOA ALT > 40 IU/1 Int Arch Occup Environ Health I---------------------------- -U-------------------------------- In PFOA Fig. 6 Scatterplots of the natural log PFOA (ng/ml) by the natural log T4 and free T4 fasting requirements for blood collection, including caffeinated drinks. It can also be hypothesized that the associ ations observed for serum triglycerides and PFOA might be the consequence of the non-adherence to fasting require ments by some shift production workers and/or the effect of postprandial metabolic responses in shift workers. Sev eral studies have indicated postprandial serum triglyceride levels are higher among night shift workers than day workers (Al-Naimi et al. 2004; Morgan et al. 1998; Karlsson et al. 2001 ; Lund et al. 2001). If a subset of subjects (i.e., production workers with higher PFOA serum con centrations) who worked night shift were less likely to adhere to the fasting requirements and/or have postprandial metabolic profiles similar to other night shift workers, then a non-causal positive association between PFOA concen trations and serum triglycerides could be observed when all subjects are included in the analysis. Unfortunately, at the time of data blood collection shift status information was not obtained; consequently with this database it is not possible to further address this methodological, question. Countering this possible hypothesis is the fact that blood glucose, also requiring a fasting-sample, was not associated with PFOA at any site. However, hyperglycemia has not been consistently associated with night shift workers (Al-Naimi et al. 2004; Karlsson et al. 2003). Also, the association between serum triglycerides and PFOA was not Table 11 Non-adjusted and adjusted natural log (In) PFOA coefficients with In thyroidrelated hormone measurements " See study methods. Adjusted for Ln age, Ln BMI, Ln alcohol Non-adjusted Ln PFOA Coefficient Ln TSH All locations 0.0395 Antwerp 0.0509 Cottage Grove : -0.0016 Decatur 0.0343 Ln T4 All locations -0.0037 Antwerp -0.0022 Cottage Grove -0.0124 Decatur -0.0012 Ln free T4 All locations -0.0138 Antwerp -0.0108 Cottage Grove -0.0093 Decatur -0.0138 Ln T3 All locations 0.0107 Antwerp 0.0222 Cottage Grove 0.0026 Decatur 0.0317 SE 0.0204 0.0329 0.0310 0.0497 0.0054 .0099 0.0072 0.0126 0.0044 0.0078 0.0058 0.0103 0.0052 0.0077 0.0096 0.0117 P value 0.05 0.12 0.96 0.49 0.50 0.83 0.09 0.92 0.002 0.17 0.11 0.18 0.04 0.005 0.79 0.008 Adjusted" Ln PFOA Coefficient 0.0360 0.0391 -0.0111 0.0365 -0.0057 -0.0041 -0.0093 -0.0083 -0.0117 -0.0140 -0.0071 -0.0184 0.0105 0.0216 0.0006 0.0271 SE 0.0207 0.0333 0.0322 0.0513 0.0054 0.0099 0.0072 0.0127 0.0043 0.0078 0.0059 0.0105 0.0053 0.0077 0.0099 0.0119 P value 0.08 0.24 0.73 0.48 0.29 0.68 0.20 0.51 0.01 0.07 0.23 0.08 0.05 0.006 0.95 0.02 Springer Int Arch Occup Environ Health Table 12 Predicted thyroid-related measurements (reference range in parentheses) based on regression models for 40 year-old male with BMI = 28, and consumes 0.5 alcoholic drinks per day PFOA (pg/ml) Predicted TSH (iiIU/ml) (0.25-5.5) Predicted T4 (pg/dl) (4.5-12.0) Predicted free T4 (ng/dl) (0.70-1.53) Predicted T3 (ng/dl) (60-181) 0.005 0.01 0.10 0.50 1.00 5.00 10.00 50.00 100.00 1.58 1.62 1.76 1.87 1.91 2.03 2.07 2.20 2.26 8.22 1.15 8.19 1.15 8.08 1.11 8.01 1.09 7.98 1.09 7.90 1.06 7.87 1.06 7.80 1.04 7.77 1.03 119 119 121 124 125 128 128 131 132 observed at the Cottage Grove site which had both pro duction and non-production workers. Nevertheless, a po sitive association between serum triglycerides and PFOA was reported by Kaplan (2004) whose study population also consisted of production (shift workers) and non-pro duction (non-shift workers) participants. To further clarify a possible positive association, between workers' PFOA concentrations and serum triglyceride levels, adjustment for shift work becomes a methodological necessity for subsequent occupational research analyses. Although hepatic enzymes, in particular GGT, AST and ALT are known to be elevated with heavy alcohol con sumption, these liver enzymes can also be elevated due to obesity and dyslipidemia (Collantes et al. 2004; Mofrad and Sanyal 2003; Ruhl and Everhart 2003) as non-alco holic fatty liver disease has substantially increased in prevalence in the US population, as indicated by the third National Health and Nutrition Examination Survey (NHANES) (Clark et al. 2003). Measured hepatic clinical chemistry enzymes in the present study were not consis tently associated with employees' serum PFOA concen trations adjusted for age, alcohol, and either BMI or serum triglycerides. A weakly positive asscciation between alkaline phosphatase, ALT and GGT with PFOA was ob served among the Decatur male participants. Several epi demiologic research studies of the Decatur workforce have not reported associations with liver disease (malignant or non-malignant conditions) using a variety of data sources including death certificates (Alexander et al. 2003), epi sodes of care (Olsen et al. 2004) and self-reports (Alex ander and Grice 2006). Neither a worker population study (Kaplan 2004) nor a community-based PFOA-exposed population (Emmett et al. 2006a, b) has reported associa tions between PFOA and hepatic clinical chemistries. In the Emmett et al. (2006a) study, there was no relationship between the blood levels of PFOA and the results for cholesterol, hepatic-related tests (serum protein, albumin, bilirubin, serum alkaline phosphatase, AST, ALT and GGT), or being treated for or informed by a physician that a community participant had liver disease (cirrhosis, hep atitis, and any other liver condition). In the present study, there, were no consistent associa tions between TSH, T4, free T4 or T3 with PFOA across the individual facility locations. Overall, T4 was negatively associated with PFOA and T3 was positively associated but these trends were well within normal reference ranges. The lack of thyroid related hormone associations is also con sistent with results from Kaplan (2004) and Emmett et al. (2006a). In a 6-month oral capsule PFOA dose study of cynomolgus monkeys, Butenhoff et al. (2002) reported no Table 13 Adjusted mean and 95% confidence intervals for thyroid measurements, by PFOA decile PFOA Decile TSH Mean 95% Cl T4 Mean 95% Cl Free T4 Mean 95% Cl 1 2.07d 1.13-3.01 8.29 7.92-8.66 1.15e,gJ 1.11-1.19 2 2.00d 1.04-2.96 8.45 8.07-8.83 1.11 1.07-1.15 3 1.89d 0.94-2.84 8.08 7.70-8.46 1.14eJ 1.10-1.18 4 3.43a_c,r 2.48-4.38 8.04 7.67-8.41 1.12 1.08-1.16 5 2.61 1.65-3.55 7.98f 7.60-8.35 1.07ac 1.03-1.11 6 2.07d 1.11-3.03 8.53eJ 8.15-8.91 1.10 1.05-1.14 7 2.21 1.26-3.16 8.07 7.70-8.44 1.09" 1.06-1.14 8 2.36 1.46-3.32 8.40 8.03-8.76 1.08 1.06-1.14 9 2.84 1.87-3.81 8.42 8.03-8.80 1.11 1.06-1.15 10 2.40 1.46-3.37 7.94f 7.56-8.32 1.07'c 1.03-1.11 Adjusted for age, BMI and alcohol Statistically significantly (P < 0.05) different than PFOA decile(s) 1, 2, 3,.. .,10. respectively Springer T3 Mean 124 125 124 126 126 128 129 130 129 128 95% Cl 118-131 118-131 118-131 120-133 120-133 121-134 123-135 124-136 123-136 123-136 Int Arch Occup Environ Health clear changes in thyroid hormone homeostasis for TSH, T4 of free thyroxine in rat serum containing perfluorooctanesulfo- or free T4. Three high-dose monkeys (serum concentra tions mentioned above) that were removed from dosing due to toxicity had T3 values that trended downward compared nate (PFOS). Toxicology 234:21-33 Clark JM, Brancati FL, Diehl AM (2003) The prevalence and etiology of elevated aminotransferase levels in the United States. Am J Gastroenterol 98:960-967 to pretreatment measures. This is opposite the observation Collantes R, Ong JP, Younossi ZM (2004) Nonalcoholic fatty liver in the present study. Finally, a negative bias in thyroidrelated analog methods, using another perfluoroalkyl acid (PFOS), has recently been reported by Chang et al. (2007). disease and the epidemic of obesity. Cleve Clin J Med 71:657-664 Costa G (2004) Letter. US EPA docket AR226-1868. US Environ mental Protection Agency, Washington, DD De Silva AO, Mabury SA (2006) Isomer distribution of perfluoro- carboxylates in human blood: potential correlation to source. Environ Sci Technol 40:2903-2909 Conclusion Emmett EA, Zhang H, Shofer FS, Freeman D, Rodway NV, Desai C, Shaw LM (2006a) Community exposure to perfluorooctanoate: relationships between serum levels and certain health parame PFOA concentrations measured in this workforce of 506 male fluorochemical production workers were not associ ated with total cholesterol, LDL, hepatic enzymes or thy ters. JOEM 48:771-779 Emmett EA, Zhang H, Shofer FS, Freeman D, Rodway NV, Desai C, Shaw LM (2006b) Community exposure to perfluorooctanoate: relationships between serum concentrations and exposure roid hormones. Residual confounding likely explained an association between PFOA and HDL. Several hypotheses are offered for the inconsistent associations observed be tween PFOA and triglycerides. Although a biological ' sources." JOEM 48:759-770 Expert Panel (2001) Executive summary of the third report of the National Cholesterol Education Program (CNCEP) Expert Panel on detection, evaluation, and treatment of high blood cholestrol in adults (Adult Treatment Panel HI). JAMA-285:2486-2497 explanation cannot b niled out, other hypotheses are also' Friedewald WT, Levy RI, Fredrickson DS (1972) Estimation of the plausible. These include non-adherence to fasting require ments for morning blood collection by night shift produc -concentration of low-density lipoprotein cholesterol in plasma without use of the preparative ultracentrifuge. Clin Chem 18:499-502 tion workers and/or increased triglycride levels that have' Gilliland FD, Mandel JS ( i 996) Serum perfluorooctanoic acid and been found in night shift workers, in general. Because " hepatic enzymes, lipoproteins and cholesterol: a study of PFOA has been shown to bind or be carried to blood albumin and beta-lipoproteins in the rat, monkey and hU occupationally exposed men. Am J Ind Med 129:560-568 Han X, Snow TA, Kemper RA, Jepson GW (2003) Binding of perfluorooctanoic acid to rat and human plasma proteins. Chem man, a non-causal positive cdrrelation also remains another Res Toxicol 16:775-781 possible explanation. Hansen KJ, Clemen LA, Ellefson ME, Johnson HO (2001) Compound-specific quantitative characterization of organic References i fluprochemicals in biological matrices. Environ Sci Technol 35:766-770 ' Hernn MA, Hemndez-Diz S, Werler MM, Mitchell AA (2002) Causal knowledge as a prerequisite for confounding evaluation: Alexander BH, Grice M (2006) Self-reported medical conditions in an application to birth defects epidemiology. Am J Epidemiol perfluorooctanesulfonyl fluoride manufacturing workers. Uni 155:176-184 versity of Minnesota, Minneapolis, 1 February, 2006. US EPA Kaplan AM (2004) Ammonium perfluorooctanoate. US EPA docket docket AR-226-3677. US Environmental Protection Agency, Washington, DC AR-226-1867 and AR 226-1868. US Environmental Protection Agency, Washington, DC Alexander BH, Olsen GW, Burris JM, Mandel JH, Mande! JS (2003) Karlsson B, Knutsson A, Lindahl B (2001) Is there an association Mortality of employees of a perfluorooctanesulphonyl fluoride between shift work and having a metabolic syndrome? Results manufacturing facility. Occup Environ Med 60:722-729 Al-Naimi S, Hampton SM, Richard P, Tzung C, Morgan LM (2004) from a population based study of 27,485 people. Occup Environ Med 58:747-752 . Postprandial metabolic profiles following meals and snacks eaten Karlsson BH, Knutsson AK, Lindahl BO, Alfredsson LS (2003) during simulated night and day shift work. Chronobiol Int 21:937-947 - Metabolic disturbances in male workers with rotating three-shift work. Results of the WOLF study. Int Arch Occup Environ Brousseau ME, Schaefer EJ, Wolfe ML, Bloedon LT, Digenio AG, Health 76:424-430 Clark RW, Mancuso JP, Rader DJ (2004) Effects of an inhibitor of cholesteryl ester transfer protein on HDL cholesterol. N Engl J Med 350:1505-1515 Butenhoff J, Costa G, Elcombe C, Farrar D, Hansen K, Iwai H, Jung Kennedy GL, Butenhoff JL, Olsen GW, O'Connor JC, Seacat AM, Perkins RG, Biegel LB, Murphy SR, Farrar DG (2004) The toxicology of perfluorooctanoate. Crit Rev Toxicol 34:351-384 Kerstner-Wood C, Coward L, Gorman G (2003) Protein binding of R, Kennedy G, Lieder P, Olsen G, Thomford P (2002) Toxicity of ammonium perfluorooctanoate in male cynomolgus monkeys after oral dosing for 6 months. Toxicol Sci 69:244--257 Calafat AM, Kuklenyik Z, Caudill SP, Reidy JA, Needham LL (2006) Perfluorochemicals in pooled serum samples from United States residents in 2001 and 2002. Environ Sci Technol 40:2128-2134 perfluorobutane sulfonate, perfluorohexane sulfonate, perfluorooctane sulfonate and perfluorooctanoate to plasma (human, rat, and monkey), and various human-derived plasma protein fractions. Southern Research Institute. Study 9921.7. US EPA docket AR-226-1354. US Environmental Protection Agency, Washington, DC Chang SC, Thibodeaux JR, Eastvold ML, Ehresman DJ, Bjork JA, Lund J, Arendt J, Hampton SM, English J, Morgan LM (2001) Froehlich JW, Lau CS, Singh RJ, Wallace KB, Butenhoff JL (2007) Negative bias from analog methods used in the analysis Postprandial hormone and metabolic responses amongst shift workers in Antarctica. J Endocrinol 171:557-564 Springer p. 16 Int Arch Occup Environ Health Maloney EK, Waxman DJ (1999) Transactivation of PPARa and PPARy by structurally diverse environmental chemicals. Toxicol Appl Pharmacol 161:209-218 Mofrad PS, Sanyal AJ (2003) Nonalcoholic fatty liver disease. MedGenMed 5:1-13 Morgan L, Arendt J, Owens D, Folkard S, Hampton S, Deacon S, English J, Ribeiro D, Taylor K (1998) Effects of the endogenous clock and sleep time on melatonin, insulin, glucose, and lipid metabolism. J Endocrinol 157:443-451 Nakanishi N, Matsuo Y, Yoneda H, Nakamur K, Suzuki K, Tatara K (2000) Validity of the conventional indirect methods including Friedewald method for determining serum low-density lipopro tein cholesterol level: comparison with the direct homogeneous enzymatic analysis. J Occup Health 42:130-137 Olsen GW, Zobel LR (2006) An analysis of the 2000 fluorochemical (perfluorooctanoate, PFOA) medical surveillance program at 3M Company's Antwerp (Belgium), Cottage Grove (Minnesota), and Decatur (Alabama) facilities. 3M Company, St Paul, 6 May, 2006. US EPA docket AR-226-3678. US Environmental Pro tection Agency, Washington, DC Olsen GW, Gilliland FD, Burlew MM, Burris JM, Mandel JS, Mandel JH (1998) An epidemiologic investigation of reproductive hormones in men with occupational exposure to perfluorooctanoic acid. JOEM 40:614-622 Olsen GW, Burris JM, Mandel JH, Zobel LR (1999) Serum perfluorooctane sulfonate and hepatic and lipid clinical chem istry tests in fluorochemical production employees. JOEM 42:799-806 Olsen GW, Burris JM, Burlew MM, Mandel JH (2000) Plasma cholecystokinin and hepatic enzymes, cholesterol and lipopro teins in ammonium perfluorooctanoate production workers. Drug Chem Toxicol 23:603-620 Olsen GW, Burris JM, Burlew MM, Mandel JH (2003a) Epidemi ologic assessment of worker serum perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) concentrations and medical surveillance examinations. JOEM 45:260-270 Olsen GW, Church TR, Miller JP, Burris JM, Hansen KJ, Lundberg JK, Armitage JB, Herron RM, Medhdizadehkashi Z, Nobiletti JB, O'Neill EM, Mandel JH, Zobel LR (2003b) Perfluorooc tanesulfonate and other fluorochemicals in the serum of Amer ican Red Cross adult blood donors. Environ Health Perspect 111:1892-1901 Olsen GW, Butenhoff JL, Mandel JH (2003c) Assessment of lipid, hepatic and thyroid function in relation to an occupational biologic limit value for perfluorooctanoate. 3M Company, St Paul. US EPA docket AR-226-1351. US Environmental Protec tion Agency, Washington, DC Olsen GW, Burlew MM, Marshall JC, Burris JM, Mandel JH (2004) Analysis of episodes of care in a perfluorooctanesulfonyl fluoride production facility. JOEM 46:837-846 Ruhl CE, Everhart JE (2003) Determinants of the association of overweight with elevated serum alanine aminotransferase activ ity in the United States. Gastroenterology 124:71-79 Seacat AM, Thomford PJ, Hansen KJ, Olsen GW, Case MT, Butenhoff JL (2002) Subchronic toxicity studies on perfluorooc tanesulfonate potassium salt in cynomolgus monkeys. Toxicol Sei 68:249-264 Seacat AM, Thomford PJ, Hansen KJ, Clemens LA, Eldridge SR, Elcombe CR, Butenhoff JL (2003) Sub-chronic dietary toxicity of potassium perfluorooctanesulfonate in rats. Toxicology 183:117-131 Stein EA, Myers GL (1994) Lipids, lipoproteins, and apolipoproteins. In: Burtis CA, Ashwood ER (eds) Tietz textbook of clinical chemistry. WB Saunders Co., Philadelphia, pp 1002-1093 Ubel FA, Sorenson SD, Roach DE (1980) Health status of plant workers exposed to fluorochemicals--a preliminary report. Am Ind Hyg Assoc J 41:584--589 Xie Y, Yang Q, Nelson BD, DePierre JW (2003) The relationship between liver peroxisome proliferation and adipose tissue atrophy induced by peroxisome proliferators exposure and withdrawal in mice. Biochem Pharmacol 66:749-756 " Springer