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Albumin is the major carrier protein for PFOS, PFOA, PFHxS, PFNA and PFDA in human plasma
Environment International 137 (2020) 105324
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Albumin is the major carrier protein for PFOS, PFOA, PFHxS, PFNA and PFDA in human plasma
T
Martin Forsthubera,b, Andreas Marius Kaiserb,c, Sebastian Granitzerb, Ingrid Hassld, ⁎ Markus Hengstschlägerb, Herbert Stangld, Claudia Gundackerb, a
Department of Environmental Health, Center for Public Health, Medical University of Vienna, A-1090 Vienna, Austria Institute of Medical Genetics, Center for Pathobiochemistry and Genetics, Medical University of Vienna, A-1090 Vienna, Austria c Environment Agency Austria, Spittelauer Lände 5, A-1090 Vienna, Austria d Institute of Medical Chemistry, Center for Pathobiochemistry and Genetics, Medical University of Vienna, A-1090 Vienna, Austria b
A R T I C LE I N FO
A B S T R A C T
Handling Editor: Adrian Covaci
Perfluoroalkyl (PFAS) substances are widespread in the environment and in organisms. The fact that exposure to PFAS is associated with elevated cholesterol levels is a major concern for human health. Previous investigations, in which bovine serum albumin was frequently studied, indicate that PFOS, PFOA and PFNA bind to serum albumin. However, it is critical to know whether these and other PFAS have a preference for the protein or the lipid fraction in native human blood fractions. For this reason, blood samples from four young healthy volunteers (two women, two men, 23–31 years old) were used for protein size separation and fractionation by the Cohn method in combination with serial ultracentrifugation. The plasma fractions were analyzed for 11 PFAS using high-performance tandem mass spectrometry (HPLC-MS/MS). Although the data are based on a small sample, they clearly show that albumin is the most important carrier protein for PFOS, PFOA, PFHxS, PFNA and PFDA in native human plasma. These five compounds have very little or no affinity for lipoproteins. The confirmation of their transport through albumin is important for the epidemiology of PFAS. The present results must be verified by the examination of a larger number of persons.
Keywords: Albumin Human plasma perfluoroalkyl substances (PFAS)
1. Introduction Perfluoroalkyl substances (PFAS) are a class of industrial chemicals, which are produced in a variety of industries around the globe since decades. PFAS are employed in manufacturing processes of everyday life products such as fat repellent food packaging or waterproof fabrics. There are more than 4,700 different compounds registered worldwide (OECD, 2018) and their numbers are rapidly increasing. Numerous PFAS are considered as highly persistent in the environment and can be detected in water and soil (Kabore et al., 2018; Rankin et al., 2016). Perfluorooctansulfonate (PFOS) and perfluorooctanoic acid (PFOA) are the most frequent PFAS present in the environment and in humans (Buck et al., 2011; Calafat et al., 2007; Kannan et al., 2004). The human health concerns arising from PFAS exposure are manifold. Adverse
outcomes such as increased serum cholesterol levels, immunological effects and reduced birth weight are of concern (Chang et al., 2016; Govarts et al., 2018; Grandjean et al., 2017; Steenland et al., 2009). There are clear differences between mammalian species that make it difficult to assess causal mechanisms and their relevance for humans. For example, the half-life of PFOA and PFOS is hours to days in mice, rats and monkeys, but years in humans (Pizzurro et al., 2019). According to the European Food Safety Authority (EFSA) the median concentrations for PFOS and PFOA in human serum of European adults (sample collection period: 2007–2015) are 7.7 ng/ml and 1.9 ng/ml, respectively. Both substances are detectable in almost all individuals in Europe and are comparable to the general population worldwide (EFSA, 2018). Comparison of whole blood and plasma indicates accumulation of PFOS and PFOA in the non-cellular blood
Abbreviations: EDTA, Ethylenediaminetetraacetic acid; HDL, High density lipoproteins; HPLC-MS/MS, High-performance liquid chromatography tandem-mass spectrometry; LDL, Low density lipoproteins; PFAS, Perfluoroalkyl substances; PFBA, Perfluoro-n-butanoic acid; PFOS, Perfluorooctansulfonate; PFDA, Perfluorodecanoic acid; PFHpA, Perfluoroheptanoic acid; PFHxS, Perfluorohexanesulfonic acid; PFHxA, Perfluorohexanoic acid; PFNA, Perfluorononanoic acid; PFDoDA, Perfluorooctadecanoic acid; PFOA, Perfluorooctanoic acid; PFPeA, Perfluoropentanoic acid; PFUnDA, Perfluoroundecanoic acid; VLDL, Very low density lipoproteins; FOSA, Perfluorooctanesulfonamide ⁎ Corresponding author at: Institute of Medical Genetics, Medical University of Vienna, Währinger Strasse 10, A-1090 Vienna, Austria. E-mail address: [email protected] (C. Gundacker). https://doi.org/10.1016/j.envint.2019.105324 Received 3 September 2019; Received in revised form 4 November 2019; Accepted 11 November 2019 0160-4120/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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phases were centrifuged twice to increase purity and ensure removal of albumin. Afterwards, the isolated lipoproteins were dialyzed two times, overnight with dialysis buffer (0.9% NaCl, pH 7,4) to remove potassium bromide and with PBS to remove ethylenediaminetetraacetic acid (EDTA). For separation of the plasma samples into < 30 kD and > 30 kD compartments, centrifugal filters (Amicon® Ultra −15, −30 K) were used. Albumin was isolated from EDTA stabilized plasma using the Cohn method (Kistler and Nitschmann 1962). In short, plasma samples were acidified, cooled and centrifuged in several steps to recover highly purified albumin. Protein concentrations were determined via NanoDrop® (ND-1000, PeqLab) measurements.
fraction (Ehresman et al., 2007). Therefore, PFAS are primarily measured in human serum or plasma (Olsen et al., 2017). The binding of PFAS to plasma constituents such as albumin or lipoproteins is still a matter of discussion (addressed by USEPA in 2016 and by EFSA in 2018). On the basis of earlier, fundamental work (Beesoon and Martin 2015; Bischel et al., 2010, 2011; Butenhoff et al., 2012; Chen et al., 2015; Chi et al., 2018; Luo et al., 2012; Ng and Hungerbuehler 2015; Salvalaglio et al., 2010) it can be assumed that albumin is the most likely ligand. In addition, structural similarities to fatty acids indicate a corresponding transport via the blood stream (van der Vusse 2009). Normally, blood fatty acids are transported in the free form as a constituent of albumin but also in lipoproteins mainly as part of triglycerides and cholesteryl ester. Besides, there are plenty of other serum proteins (e.g., hormone-binding globulins, Jones et al., 2003) that could interact with PFAS. Since we hypothesize that PFAS behave like free fatty acids with regard to their kinetics, we have limited our investigation to their primary carriers. In accordance with the literature (Ehresman et al., 2007) and based on our own experience in fatty acid metabolism, we hypothesized PFAS to be present mainly in the non-cellular phase of blood. To identify the major ligand of PFAS in human blood of young volunteers (two women, two men), we analyzed the concentrations of 11 perfluoroalkyl acids (PFAAs) (perfluoro-n-butanoic acid (PFBA), perfluorooctansulfonate (PFOS), perfluorodecanoic acid (PFDA), perfluoroheptanoic acid (PFHpA), perfluorohexanesulfonic acid (PFHxS), perfluorohexanoic acid (PFHxA), perfluorononanoic acid (PFNA), perfluorooctadecanoic acid (PFDoDA), perfluorooctanoic acid (PFOA), perfluoropentanoic acid (PFPeA) and perfluoroundecanoic acid (PFUnDA)) in size separated plasma samples (> 30 kD, < 30 kD), very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL) as well as in an albumin fraction known to carry free fatty acids.
2.2. PFAS analyses The simultaneous determination of PFBA, PFPeA, PFHxA, PFHxS, PFHpA, PFOA, PFOS, PFNA, PFDA, PFUnDA and PFDoDA was performed at the Environment Agency Austria by high-performance liquid chromatography tandem-mass spectrometry (HPLC-MS/MS). Plasma samples as well as the plasma fractions > 30 kD and < 30 kD were prepared using a modified method published by Kuklenyik et al., (2004). The samples containing lipoproteins (VLDL, LDL and HDL) were mixed with 150 µl of 0.5 M formic acid (HFA), 5 mL acetonitrile (ACN) and 2 mL n-hexane. The lipoprotein-mixtures were roughly shaken, vortexed and put in an ultrasonic bath for 20 min. After centrifugation the n-hexane supernatant was discarded, and the remaining ACN was cleaned up with 100 mg of an ENVI-Carb sorbents (Supelco, Bellefonte, USA). The volumes of the samples were reduced to 1 mL under a nitrogen flow and by heating the samples up to 40 °C. The liquid part of the albumin samples was prepared equal to the plasma method and for the solid albumin pellet the same method as for the lipoproteins were applied without the n-hexane. Before the sample preparation all samples were spiked with isotope-labelled standards. The calibration included 10 concentrations ranging between 0.05 and 25 ng/mL. Standards were purchased from Wellington Laboratories with a purity > 98%. Methanol (Pestinorm, VWR International, Leuven, Belgium) and ACN (Optigrade, Promochem, LGC Standards, Wesel, Germany) were distilled before usage. PFAS-free water was prepared by filtering tap water with Oasis HLB-column (6 cc Cartridge 200 mg) cartridges (Waters Corporation, Milford, MA, USA). An Agilent Technologies 1290 Infinity Series (Agilent Technologies, Santa Clara, CA, USA), and the MS detector system AB Applied Biosystem MDS SCIEX 400 QTRAP LC/MS/MS System (AB Sciex Technologies, Framingham, MA, USA) in electrospray ionization (ESI) negative mode were used for the PFAS analysis. The analytical column was a Luna 5 µm C18(2), 100 × 2 mm (Phenomenex, California, USA). The mean recoveries of the spiked samples were 86 ± 27% for the sera and 99 ± 12% for the lipoproteins. The limits of detection (LOD) and limits of quantification (LOQ) are given in Table 1.
2. Methods 2.1. Isolation of lipoproteins, albumin and separation into two plasma fractions (< 30 kD, > 30 kD) Whole blood samples of two healthy women (23 and 27 years of age) and two healthy men (28 and 31 years of age) were collected in Sarstedt® tubes (S-Monovette K3 EDTA) and centrifuged (3,900 RPM (Eppendorf 5810 R), 4 °C, 20 min) to obtain plasma. The blood sampling was approved by the Ethics Committee of the Medical University of Vienna (EK No. 1414/2019). Lipoproteins were isolated by serial ultracentrifugation (52,000 RPM, rotor 55.2 Ti ((Beckman Coulter®), 4 °C, 20 h)) and density adjustment with potassium bromide (VLDL 1.019 g/L KBr, LDL 1.063 g/L KBr, HDL 1.22 g/L KBr). The upper
Table 1 Limit of Detection (LOD) and Limit of Quantification (LOQ) for PFAS analyzed in plasma (pure plasma, fractions > 30 kD, < 30 kD and albumin) and in lipoproteins (VLDL, LDL and HDL). PFAS
Perfluoro-n-butanoic acid (PFBA) Perfluoropentanoic acid (PFPeA) Perfluorohexanoic acid(PFHxA) Pperfluoroheptanoic acid (PFHpA) Perluorooctanoic acid (PFOA) Perfluorononanoic acid (PFNA) Perfluorodecanoic acid (PFDA) Perfluoroundecanoic acid (PFUnDA) Perfluorododecanoic acid (PFDoDA) Perfluorohexane sulfonate (PFHxS) Perfluorooctane sulfonate (PFOS)
Plasma
Lipoproteins
LOD [ng/ml]
LOQ [ng/ml]
LOD [ng/ml]
LOQ [ng/ml]
0.125 0.225 0.040 0.045 0.10 0.065 0.0225 0.035 0.030 0.060 0.12
0.25 0.45 0.080 0.090 0.20 0.13 0.045 0.070 0.060 0.12 0.24
0.10 0.20 0.035 0.0425 0.10 0.0425 0.0225 0.0325 0.0175 0.050 0.075
0.20 0.40 0.070 0.085 0.20 0.085 0.045 0.065 0.035 0.10 0.15
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2.3. SDS-PAGE and protein staining For SDS-PAGE a 12.5% SDS-polyacrylamide gel with a thickness of 1.5 mm was poured and stored under moist conditions (+4 °C) over night. The protein samples (1.5 µl) were mixed with a loading dye (200 mM Tris pH 6.8, 400 mM DTT, 8% SDS, 0.4% Bromphenol blue, 40% Glycerol, dH2O) and gently applied to the gel. As reference a prestained protein ladder (Page Ruler™, Thermo Scientific) was used in order to identify proteins of loaded samples. The electrophoresis was performed with a Mini-Protean® Tetra Vertical Electrophoresis Cell from BioRad® at 70 Volt. Afterwards, the gel was stained for one hour with a protein staining solution (Coomassie Brilliant Blue Staining Solution R-250, BioRad®) and destained for two hours with a destaining solution (Coomassie Brilliant Blue Destaining Solution R-250, BioRad®). Then, the gel was visualized with a scanner (Samsung® M2070). 3. Results 3.1. PFAS concentrations in total plasma Out of the 11 PFAAs analyzed in plasma, PFOS, PFOA, PFHxS, PFNA and PFDA were detected in all samples (Table 2). The highest mean and maximum concentrations were found for PFOS. The PFAAs concentrations found in total plasma are comparable with the average burden reported for Europe (EFSA, 2018). The plasma concentrations are comparable with the concentrations in the albumin and the > 30 kD fraction, which is especially true for PFOS and PFNA. 3.2. PFAS concentrations in plasma size fractions
Fig. 1. Coomassie Brilliant Blue staining of a SDS-polyacrylamide gel (12.5%) loaded with 1.5 µl plasma and albumin samples. The molecular weight of albumin is ∼ 65 kDa. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Next we aimed to identify the blood fraction that shows an affinity to the substances. Therefore, size fractioning via ultrafiltration tubes was performed, to test if PFAS are freely available or bound to proteins bigger than 30 kD. In the < 30 kD fraction, the levels of all 11 PFAAs were below the respective limits of detection. Comparing PFAS contents of the > 30 kD fraction to those of total plasma indicates that the substances are predominantly bound to plasma proteins bigger than 30 kD (Table 2)
albumin. Plasma protein fractioning after Cohn (Kistler and Nitschmann 1962) is a widely used and efficient method for isolating highly purified plasma constituents such as albumin. To ensure a high purification level was reached, a protein staining was performed (Fig. 1). The albumin fraction contained the majority of PFOS, PFOA, PFHxS, PFNA and PFDA (Table 2).
3.3. PFAS concentrations in plasma lipoproteins and plasma albumin 4. Discussion
Both, the lipoproteins VLDL, LDL and HDL as well as albumin are known to carry fatty acids (van der Vusse 2009). Therefore, we separated the lipoproteins by density gradient centrifugation. To ensure the purity, we depleted other plasma constituents by an additional second spin. This means in effect that impurities (other plasma constituents such as albumin) are reduced in the LDL fraction and highly reduced in the HDL fraction. In the overall lipoprotein fraction we could detect PFOS, PFNA and PFDA, however, the amount was very low compared to plasma (Table 2). Surprisingly, the highest PFAAs concentrations in the lipoprotein fraction were detected in HDL, which is considered as the fraction with the highest purity. Finally we used a well-established method to isolate highly purified
Our findings show that not only PFOS, PFOA and PFNA have a high affinity to the albumin fraction, but also PFHxS and PFDA. We have determined the distribution of these five PFAS compounds in native blood fractions. The five compounds differ in chain length and include the most common functional groups (sulfonyl and carboxylic acids), indicating that the finding might also apply to other PFAS. The confirmation of their transport through albumin is important for the interpretation of epidemiology studies on PFAS. The present results must therefore be verified by the examination of a larger number of persons. This is necessary in order to better interpret the relatively
Table 2 Concentrations (ng/mL) of PFOS, PFOA, PFHxS, PFNA and PFDA in total plasma and plasma fractions of two female and two male participants. Values represent the arithmetic mean and standard deviation. Values in parentheses represent the range.
Plasma Albumin > 30 kD < 30 kD VLDL LDL HDL
PFOS
PFOA
PFHxS
PFNA
PFDA
4.3 ± 2.9 (1.7–8.4) 4.3 ± 2.2 (3.3–4.7) 3.5 ± 3.1 (1.0–7.9) < LOD < LOD 0.1 ± 0.1 (0.06–0.3) 0.16 ± 0.06 (0.1–0.2)
1.9 ± 0.8 (0.9–2.7) 2.8 ± 0.9 (2.0–4.1) 1.3 ± 0.6 (0.4–1.9) < LOD < LOD < LOD < LOQ
0.9 ± 0.9 (0.3–2.3) 1.4 ± 1.0 (0.7–2.8) 0.6 ± 0.5 (0.1–1.4) < LOD < LOD < LOD < LOD
0.5 ± 0.1 (0.4–0.6) 0.6 ± 0.3 (LOD–0.7) 0.4 ± 0.2 (0.2–0.6) < LOD < LOD < LOD 0.01 ± 0.01 (LOD–0.02)
0.3 ± 0.1 (0.1–0.4) 0.5 ± 0.2 (LOQ–0.7) 0.2 ± 0.1 (0.08–0.3) < LOD < LOD < LOQ 0.01 ± 0.004 (LOD–0.009)
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Acknowledgements
high variance of concentrations in serum and serum fractions observed here and also to detect possible sex-specific differences. Our focus on proteins known to bind free fatty acids is another limitation of the study. Our approach may have overlooked less abundant proteins that can bind PFAS with high affinity. Our data, however, are very consistent with the results from previous studies, which showed that the majority of PFOS and PFOA was found in lipoprotein-poor fractions (Butenhoff et al., 2012), that PFOS and PFOA bind to fatty acid binding sites (Salvalaglio et al., 2010), that PFOS binds more strongly to albumin than PFOA (Chi et al., 2018) and that PFNA also has a very high affinity to human albumin (Bischel et al., 2010). The observation that albuminuria is associated with increased excretion of PFAAs (Jain and Ducatman 2019) also supports our results. In addition, a comparable binding pattern of PFOA to plasma proteins was described in an experimental setting where PFOA was added to the serum of monkey, rat and human (Kerstner-Wood et al., 2003). The USEPA (2016) concluded that albumin is the primary binding protein of PFOA in plasma. While there is some evidence that PFAS are commonly found in the non-cellular phase of human blood (Ehresman et al., 2007), other reports indicate that PFHxA, PFUnDA, and FOSA are found in whole blood rather than in serum or plasma (Hanssen et al., 2013; Jin et al., 2016). However, the partly contradictory results regarding the recovery rates of PFUnDA in blood and plasma may indicate a possible loss of the compounds during separation of non-cellular and cellular compartments from whole blood. There is an ongoing discussion why PFAS are so persistent in humans (Olsen et al., 2007). The bile acid reabsorption hypothesis is based on findings indicating that PFAS and bile salts have similar uptake mechanisms. The membrane transporters OATP, Na+/taurocholate co-transporting polypeptide (NTCP) and apical sodium-dependent bile salt transporter (ASBT) are involved in the uptake of both substances (Pellicoro and Faber 2007; Zhao et al., 2015; Zhao et al., 2017). Indeed, the circulation of PFAS in the enterohepatic circle could explain the comparably long half-life in humans. However, our data suggest that only small amounts of PFAAs are attached to cholesterolcarrying proteins. Furthermore, only LDL and HDL fractions were containing PFAAs concentrations above the detection limit. PFAAs could not be detected in VLDL fractions indicating that incorporation into the lipoproteins is rather unlikely. The question of whether PFAS exposures increase cholesterol levels is much discussed. The PFAAs investigated here accumulate preferentially in the albumin fraction. However, it seems premature to conclude that the relationship between PFAS and cholesterol levels is an artifact. To answer this question further investigation of the underlying mechanisms are necessary. The structural similarities between PFAS and fatty acids could be the important commonality to understand the distribution and the bio-accumulative character of the compounds. The accumulation of PFOS, PFOA, PFHxS, PFNA and PFDA in the albumin fraction of human plasma let us conclude they are bound in blood and transferred through the body by similar mechanisms as free fatty acids. This indicates that fatty acid membrane transporter could be the relevant cellular (uptake) transporters.
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Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 4
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perfluorooctane sulfonic acid and perfluorooctanoic acid in food. EFSA J 16 (12). https://doi.org/10.2903/j.efsa.2018.5194. Kuklenyik, Z., Reich, J.A., Tully, J.S., Needham, L.L., Calafat, A.M., 2004. Automated solid-phase extraction and measurement of perfluorinated organic acids and amides in human serum and milk. Environ. Sci. Technol. 38, 3698–3704. Luo, Z.P., Shi, X.L., Hu, Q., Zhao, B., Huang, M.D., 2012. Structural evidence of perfluorooctane sulfonate transport by human serum albumin. Chem. Res. Toxicol. 25, 990–992. Ng, C.A., Hungerbuehler, K., 2015. Exploring the use of molecular docking to identify bioaccumulative perfluorinated alkyl acids (PFAAs). Environ. Sci. Technol. 49, 12306–12314. OECD, 2018. Summary Report on Updating the OECD 2007 List of Per- and Polyfluoroalkyl Substances 2018, May 4. Olsen, G.W., Burris, J.M., Ehresman, D.J., Froehlich, J.W., Seacat, A.M., Butenhoff, J.L., Zobel, L.R., 2007. Half-life of serum elimination of perfluorooctanesulfonate, perfluorohexanesulfonate, and perfluorooctanoate in retired fluorochemical production workers. Environ. Health Persp. 115, 1298–1305. Olsen, G.W., Mair, D.C., Lange, C.C., Harrington, L.M., Church, T.R., Goldberg, C.L., Herron, R.M., Hanna, H., Nobiletti, J.B., Rios, J.A., Reagen, W.K., Ley, C.A., 2017. Per- and polyfluoroalkyl substances (PFAS) in American Red Cross adult blood donors, 2000–2015. Environ. Res. 157, 87–95. Pellicoro, A., Faber, K.N., 2007. Review article: the function and regulation of proteins involved in bile salt biosynthesis and transport. Aliment Pharm. Therap. 26, 149–160.
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Albumin is the major carrier protein for PFOS, PFOA, PFHxS, PFNA and PFDA in human plasma
T
Martin Forsthubera,b, Andreas Marius Kaiserb,c, Sebastian Granitzerb, Ingrid Hassld, ⁎ Markus Hengstschlägerb, Herbert Stangld, Claudia Gundackerb, a
Department of Environmental Health, Center for Public Health, Medical University of Vienna, A-1090 Vienna, Austria Institute of Medical Genetics, Center for Pathobiochemistry and Genetics, Medical University of Vienna, A-1090 Vienna, Austria c Environment Agency Austria, Spittelauer Lände 5, A-1090 Vienna, Austria d Institute of Medical Chemistry, Center for Pathobiochemistry and Genetics, Medical University of Vienna, A-1090 Vienna, Austria b
A R T I C LE I N FO
A B S T R A C T
Handling Editor: Adrian Covaci
Perfluoroalkyl (PFAS) substances are widespread in the environment and in organisms. The fact that exposure to PFAS is associated with elevated cholesterol levels is a major concern for human health. Previous investigations, in which bovine serum albumin was frequently studied, indicate that PFOS, PFOA and PFNA bind to serum albumin. However, it is critical to know whether these and other PFAS have a preference for the protein or the lipid fraction in native human blood fractions. For this reason, blood samples from four young healthy volunteers (two women, two men, 23–31 years old) were used for protein size separation and fractionation by the Cohn method in combination with serial ultracentrifugation. The plasma fractions were analyzed for 11 PFAS using high-performance tandem mass spectrometry (HPLC-MS/MS). Although the data are based on a small sample, they clearly show that albumin is the most important carrier protein for PFOS, PFOA, PFHxS, PFNA and PFDA in native human plasma. These five compounds have very little or no affinity for lipoproteins. The confirmation of their transport through albumin is important for the epidemiology of PFAS. The present results must be verified by the examination of a larger number of persons.
Keywords: Albumin Human plasma perfluoroalkyl substances (PFAS)
1. Introduction Perfluoroalkyl substances (PFAS) are a class of industrial chemicals, which are produced in a variety of industries around the globe since decades. PFAS are employed in manufacturing processes of everyday life products such as fat repellent food packaging or waterproof fabrics. There are more than 4,700 different compounds registered worldwide (OECD, 2018) and their numbers are rapidly increasing. Numerous PFAS are considered as highly persistent in the environment and can be detected in water and soil (Kabore et al., 2018; Rankin et al., 2016). Perfluorooctansulfonate (PFOS) and perfluorooctanoic acid (PFOA) are the most frequent PFAS present in the environment and in humans (Buck et al., 2011; Calafat et al., 2007; Kannan et al., 2004). The human health concerns arising from PFAS exposure are manifold. Adverse
outcomes such as increased serum cholesterol levels, immunological effects and reduced birth weight are of concern (Chang et al., 2016; Govarts et al., 2018; Grandjean et al., 2017; Steenland et al., 2009). There are clear differences between mammalian species that make it difficult to assess causal mechanisms and their relevance for humans. For example, the half-life of PFOA and PFOS is hours to days in mice, rats and monkeys, but years in humans (Pizzurro et al., 2019). According to the European Food Safety Authority (EFSA) the median concentrations for PFOS and PFOA in human serum of European adults (sample collection period: 2007–2015) are 7.7 ng/ml and 1.9 ng/ml, respectively. Both substances are detectable in almost all individuals in Europe and are comparable to the general population worldwide (EFSA, 2018). Comparison of whole blood and plasma indicates accumulation of PFOS and PFOA in the non-cellular blood
Abbreviations: EDTA, Ethylenediaminetetraacetic acid; HDL, High density lipoproteins; HPLC-MS/MS, High-performance liquid chromatography tandem-mass spectrometry; LDL, Low density lipoproteins; PFAS, Perfluoroalkyl substances; PFBA, Perfluoro-n-butanoic acid; PFOS, Perfluorooctansulfonate; PFDA, Perfluorodecanoic acid; PFHpA, Perfluoroheptanoic acid; PFHxS, Perfluorohexanesulfonic acid; PFHxA, Perfluorohexanoic acid; PFNA, Perfluorononanoic acid; PFDoDA, Perfluorooctadecanoic acid; PFOA, Perfluorooctanoic acid; PFPeA, Perfluoropentanoic acid; PFUnDA, Perfluoroundecanoic acid; VLDL, Very low density lipoproteins; FOSA, Perfluorooctanesulfonamide ⁎ Corresponding author at: Institute of Medical Genetics, Medical University of Vienna, Währinger Strasse 10, A-1090 Vienna, Austria. E-mail address: [email protected] (C. Gundacker). https://doi.org/10.1016/j.envint.2019.105324 Received 3 September 2019; Received in revised form 4 November 2019; Accepted 11 November 2019 0160-4120/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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phases were centrifuged twice to increase purity and ensure removal of albumin. Afterwards, the isolated lipoproteins were dialyzed two times, overnight with dialysis buffer (0.9% NaCl, pH 7,4) to remove potassium bromide and with PBS to remove ethylenediaminetetraacetic acid (EDTA). For separation of the plasma samples into < 30 kD and > 30 kD compartments, centrifugal filters (Amicon® Ultra −15, −30 K) were used. Albumin was isolated from EDTA stabilized plasma using the Cohn method (Kistler and Nitschmann 1962). In short, plasma samples were acidified, cooled and centrifuged in several steps to recover highly purified albumin. Protein concentrations were determined via NanoDrop® (ND-1000, PeqLab) measurements.
fraction (Ehresman et al., 2007). Therefore, PFAS are primarily measured in human serum or plasma (Olsen et al., 2017). The binding of PFAS to plasma constituents such as albumin or lipoproteins is still a matter of discussion (addressed by USEPA in 2016 and by EFSA in 2018). On the basis of earlier, fundamental work (Beesoon and Martin 2015; Bischel et al., 2010, 2011; Butenhoff et al., 2012; Chen et al., 2015; Chi et al., 2018; Luo et al., 2012; Ng and Hungerbuehler 2015; Salvalaglio et al., 2010) it can be assumed that albumin is the most likely ligand. In addition, structural similarities to fatty acids indicate a corresponding transport via the blood stream (van der Vusse 2009). Normally, blood fatty acids are transported in the free form as a constituent of albumin but also in lipoproteins mainly as part of triglycerides and cholesteryl ester. Besides, there are plenty of other serum proteins (e.g., hormone-binding globulins, Jones et al., 2003) that could interact with PFAS. Since we hypothesize that PFAS behave like free fatty acids with regard to their kinetics, we have limited our investigation to their primary carriers. In accordance with the literature (Ehresman et al., 2007) and based on our own experience in fatty acid metabolism, we hypothesized PFAS to be present mainly in the non-cellular phase of blood. To identify the major ligand of PFAS in human blood of young volunteers (two women, two men), we analyzed the concentrations of 11 perfluoroalkyl acids (PFAAs) (perfluoro-n-butanoic acid (PFBA), perfluorooctansulfonate (PFOS), perfluorodecanoic acid (PFDA), perfluoroheptanoic acid (PFHpA), perfluorohexanesulfonic acid (PFHxS), perfluorohexanoic acid (PFHxA), perfluorononanoic acid (PFNA), perfluorooctadecanoic acid (PFDoDA), perfluorooctanoic acid (PFOA), perfluoropentanoic acid (PFPeA) and perfluoroundecanoic acid (PFUnDA)) in size separated plasma samples (> 30 kD, < 30 kD), very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL) as well as in an albumin fraction known to carry free fatty acids.
2.2. PFAS analyses The simultaneous determination of PFBA, PFPeA, PFHxA, PFHxS, PFHpA, PFOA, PFOS, PFNA, PFDA, PFUnDA and PFDoDA was performed at the Environment Agency Austria by high-performance liquid chromatography tandem-mass spectrometry (HPLC-MS/MS). Plasma samples as well as the plasma fractions > 30 kD and < 30 kD were prepared using a modified method published by Kuklenyik et al., (2004). The samples containing lipoproteins (VLDL, LDL and HDL) were mixed with 150 µl of 0.5 M formic acid (HFA), 5 mL acetonitrile (ACN) and 2 mL n-hexane. The lipoprotein-mixtures were roughly shaken, vortexed and put in an ultrasonic bath for 20 min. After centrifugation the n-hexane supernatant was discarded, and the remaining ACN was cleaned up with 100 mg of an ENVI-Carb sorbents (Supelco, Bellefonte, USA). The volumes of the samples were reduced to 1 mL under a nitrogen flow and by heating the samples up to 40 °C. The liquid part of the albumin samples was prepared equal to the plasma method and for the solid albumin pellet the same method as for the lipoproteins were applied without the n-hexane. Before the sample preparation all samples were spiked with isotope-labelled standards. The calibration included 10 concentrations ranging between 0.05 and 25 ng/mL. Standards were purchased from Wellington Laboratories with a purity > 98%. Methanol (Pestinorm, VWR International, Leuven, Belgium) and ACN (Optigrade, Promochem, LGC Standards, Wesel, Germany) were distilled before usage. PFAS-free water was prepared by filtering tap water with Oasis HLB-column (6 cc Cartridge 200 mg) cartridges (Waters Corporation, Milford, MA, USA). An Agilent Technologies 1290 Infinity Series (Agilent Technologies, Santa Clara, CA, USA), and the MS detector system AB Applied Biosystem MDS SCIEX 400 QTRAP LC/MS/MS System (AB Sciex Technologies, Framingham, MA, USA) in electrospray ionization (ESI) negative mode were used for the PFAS analysis. The analytical column was a Luna 5 µm C18(2), 100 × 2 mm (Phenomenex, California, USA). The mean recoveries of the spiked samples were 86 ± 27% for the sera and 99 ± 12% for the lipoproteins. The limits of detection (LOD) and limits of quantification (LOQ) are given in Table 1.
2. Methods 2.1. Isolation of lipoproteins, albumin and separation into two plasma fractions (< 30 kD, > 30 kD) Whole blood samples of two healthy women (23 and 27 years of age) and two healthy men (28 and 31 years of age) were collected in Sarstedt® tubes (S-Monovette K3 EDTA) and centrifuged (3,900 RPM (Eppendorf 5810 R), 4 °C, 20 min) to obtain plasma. The blood sampling was approved by the Ethics Committee of the Medical University of Vienna (EK No. 1414/2019). Lipoproteins were isolated by serial ultracentrifugation (52,000 RPM, rotor 55.2 Ti ((Beckman Coulter®), 4 °C, 20 h)) and density adjustment with potassium bromide (VLDL 1.019 g/L KBr, LDL 1.063 g/L KBr, HDL 1.22 g/L KBr). The upper
Table 1 Limit of Detection (LOD) and Limit of Quantification (LOQ) for PFAS analyzed in plasma (pure plasma, fractions > 30 kD, < 30 kD and albumin) and in lipoproteins (VLDL, LDL and HDL). PFAS
Perfluoro-n-butanoic acid (PFBA) Perfluoropentanoic acid (PFPeA) Perfluorohexanoic acid(PFHxA) Pperfluoroheptanoic acid (PFHpA) Perluorooctanoic acid (PFOA) Perfluorononanoic acid (PFNA) Perfluorodecanoic acid (PFDA) Perfluoroundecanoic acid (PFUnDA) Perfluorododecanoic acid (PFDoDA) Perfluorohexane sulfonate (PFHxS) Perfluorooctane sulfonate (PFOS)
Plasma
Lipoproteins
LOD [ng/ml]
LOQ [ng/ml]
LOD [ng/ml]
LOQ [ng/ml]
0.125 0.225 0.040 0.045 0.10 0.065 0.0225 0.035 0.030 0.060 0.12
0.25 0.45 0.080 0.090 0.20 0.13 0.045 0.070 0.060 0.12 0.24
0.10 0.20 0.035 0.0425 0.10 0.0425 0.0225 0.0325 0.0175 0.050 0.075
0.20 0.40 0.070 0.085 0.20 0.085 0.045 0.065 0.035 0.10 0.15
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2.3. SDS-PAGE and protein staining For SDS-PAGE a 12.5% SDS-polyacrylamide gel with a thickness of 1.5 mm was poured and stored under moist conditions (+4 °C) over night. The protein samples (1.5 µl) were mixed with a loading dye (200 mM Tris pH 6.8, 400 mM DTT, 8% SDS, 0.4% Bromphenol blue, 40% Glycerol, dH2O) and gently applied to the gel. As reference a prestained protein ladder (Page Ruler™, Thermo Scientific) was used in order to identify proteins of loaded samples. The electrophoresis was performed with a Mini-Protean® Tetra Vertical Electrophoresis Cell from BioRad® at 70 Volt. Afterwards, the gel was stained for one hour with a protein staining solution (Coomassie Brilliant Blue Staining Solution R-250, BioRad®) and destained for two hours with a destaining solution (Coomassie Brilliant Blue Destaining Solution R-250, BioRad®). Then, the gel was visualized with a scanner (Samsung® M2070). 3. Results 3.1. PFAS concentrations in total plasma Out of the 11 PFAAs analyzed in plasma, PFOS, PFOA, PFHxS, PFNA and PFDA were detected in all samples (Table 2). The highest mean and maximum concentrations were found for PFOS. The PFAAs concentrations found in total plasma are comparable with the average burden reported for Europe (EFSA, 2018). The plasma concentrations are comparable with the concentrations in the albumin and the > 30 kD fraction, which is especially true for PFOS and PFNA. 3.2. PFAS concentrations in plasma size fractions
Fig. 1. Coomassie Brilliant Blue staining of a SDS-polyacrylamide gel (12.5%) loaded with 1.5 µl plasma and albumin samples. The molecular weight of albumin is ∼ 65 kDa. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Next we aimed to identify the blood fraction that shows an affinity to the substances. Therefore, size fractioning via ultrafiltration tubes was performed, to test if PFAS are freely available or bound to proteins bigger than 30 kD. In the < 30 kD fraction, the levels of all 11 PFAAs were below the respective limits of detection. Comparing PFAS contents of the > 30 kD fraction to those of total plasma indicates that the substances are predominantly bound to plasma proteins bigger than 30 kD (Table 2)
albumin. Plasma protein fractioning after Cohn (Kistler and Nitschmann 1962) is a widely used and efficient method for isolating highly purified plasma constituents such as albumin. To ensure a high purification level was reached, a protein staining was performed (Fig. 1). The albumin fraction contained the majority of PFOS, PFOA, PFHxS, PFNA and PFDA (Table 2).
3.3. PFAS concentrations in plasma lipoproteins and plasma albumin 4. Discussion
Both, the lipoproteins VLDL, LDL and HDL as well as albumin are known to carry fatty acids (van der Vusse 2009). Therefore, we separated the lipoproteins by density gradient centrifugation. To ensure the purity, we depleted other plasma constituents by an additional second spin. This means in effect that impurities (other plasma constituents such as albumin) are reduced in the LDL fraction and highly reduced in the HDL fraction. In the overall lipoprotein fraction we could detect PFOS, PFNA and PFDA, however, the amount was very low compared to plasma (Table 2). Surprisingly, the highest PFAAs concentrations in the lipoprotein fraction were detected in HDL, which is considered as the fraction with the highest purity. Finally we used a well-established method to isolate highly purified
Our findings show that not only PFOS, PFOA and PFNA have a high affinity to the albumin fraction, but also PFHxS and PFDA. We have determined the distribution of these five PFAS compounds in native blood fractions. The five compounds differ in chain length and include the most common functional groups (sulfonyl and carboxylic acids), indicating that the finding might also apply to other PFAS. The confirmation of their transport through albumin is important for the interpretation of epidemiology studies on PFAS. The present results must therefore be verified by the examination of a larger number of persons. This is necessary in order to better interpret the relatively
Table 2 Concentrations (ng/mL) of PFOS, PFOA, PFHxS, PFNA and PFDA in total plasma and plasma fractions of two female and two male participants. Values represent the arithmetic mean and standard deviation. Values in parentheses represent the range.
Plasma Albumin > 30 kD < 30 kD VLDL LDL HDL
PFOS
PFOA
PFHxS
PFNA
PFDA
4.3 ± 2.9 (1.7–8.4) 4.3 ± 2.2 (3.3–4.7) 3.5 ± 3.1 (1.0–7.9) < LOD < LOD 0.1 ± 0.1 (0.06–0.3) 0.16 ± 0.06 (0.1–0.2)
1.9 ± 0.8 (0.9–2.7) 2.8 ± 0.9 (2.0–4.1) 1.3 ± 0.6 (0.4–1.9) < LOD < LOD < LOD < LOQ
0.9 ± 0.9 (0.3–2.3) 1.4 ± 1.0 (0.7–2.8) 0.6 ± 0.5 (0.1–1.4) < LOD < LOD < LOD < LOD
0.5 ± 0.1 (0.4–0.6) 0.6 ± 0.3 (LOD–0.7) 0.4 ± 0.2 (0.2–0.6) < LOD < LOD < LOD 0.01 ± 0.01 (LOD–0.02)
0.3 ± 0.1 (0.1–0.4) 0.5 ± 0.2 (LOQ–0.7) 0.2 ± 0.1 (0.08–0.3) < LOD < LOD < LOQ 0.01 ± 0.004 (LOD–0.009)
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Acknowledgements
high variance of concentrations in serum and serum fractions observed here and also to detect possible sex-specific differences. Our focus on proteins known to bind free fatty acids is another limitation of the study. Our approach may have overlooked less abundant proteins that can bind PFAS with high affinity. Our data, however, are very consistent with the results from previous studies, which showed that the majority of PFOS and PFOA was found in lipoprotein-poor fractions (Butenhoff et al., 2012), that PFOS and PFOA bind to fatty acid binding sites (Salvalaglio et al., 2010), that PFOS binds more strongly to albumin than PFOA (Chi et al., 2018) and that PFNA also has a very high affinity to human albumin (Bischel et al., 2010). The observation that albuminuria is associated with increased excretion of PFAAs (Jain and Ducatman 2019) also supports our results. In addition, a comparable binding pattern of PFOA to plasma proteins was described in an experimental setting where PFOA was added to the serum of monkey, rat and human (Kerstner-Wood et al., 2003). The USEPA (2016) concluded that albumin is the primary binding protein of PFOA in plasma. While there is some evidence that PFAS are commonly found in the non-cellular phase of human blood (Ehresman et al., 2007), other reports indicate that PFHxA, PFUnDA, and FOSA are found in whole blood rather than in serum or plasma (Hanssen et al., 2013; Jin et al., 2016). However, the partly contradictory results regarding the recovery rates of PFUnDA in blood and plasma may indicate a possible loss of the compounds during separation of non-cellular and cellular compartments from whole blood. There is an ongoing discussion why PFAS are so persistent in humans (Olsen et al., 2007). The bile acid reabsorption hypothesis is based on findings indicating that PFAS and bile salts have similar uptake mechanisms. The membrane transporters OATP, Na+/taurocholate co-transporting polypeptide (NTCP) and apical sodium-dependent bile salt transporter (ASBT) are involved in the uptake of both substances (Pellicoro and Faber 2007; Zhao et al., 2015; Zhao et al., 2017). Indeed, the circulation of PFAS in the enterohepatic circle could explain the comparably long half-life in humans. However, our data suggest that only small amounts of PFAAs are attached to cholesterolcarrying proteins. Furthermore, only LDL and HDL fractions were containing PFAAs concentrations above the detection limit. PFAAs could not be detected in VLDL fractions indicating that incorporation into the lipoproteins is rather unlikely. The question of whether PFAS exposures increase cholesterol levels is much discussed. The PFAAs investigated here accumulate preferentially in the albumin fraction. However, it seems premature to conclude that the relationship between PFAS and cholesterol levels is an artifact. To answer this question further investigation of the underlying mechanisms are necessary. The structural similarities between PFAS and fatty acids could be the important commonality to understand the distribution and the bio-accumulative character of the compounds. The accumulation of PFOS, PFOA, PFHxS, PFNA and PFDA in the albumin fraction of human plasma let us conclude they are bound in blood and transferred through the body by similar mechanisms as free fatty acids. This indicates that fatty acid membrane transporter could be the relevant cellular (uptake) transporters.
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Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 4
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