Binding of Perfluorooctanoic Acid to Rat and Human Plasma Proteins

to serum albumin in both rat and human blood. Introduction. Man-made organic fluorochemicals are used in indus- try as polymer additives, lubricants, ...
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Chem. Res. Toxicol. 2003, 16, 775-781

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Binding of Perfluorooctanoic Acid to Rat and Human Plasma Proteins Xing Han,* Timothy A. Snow, Raymond A. Kemper, and Gary W. Jepson DuPont Haskell Laboratory for Health and Environmental Sciences, P.O. Box 50, Newark, Delaware 19714 Received January 9, 2003

Perfluorooctanoic acid (PFOA) is a commercially important organic fluorochemical and is considered to have a long half-life in human blood. In this paper, PFOA binding to rat and human plasma proteins was investigated. On the basis of results from size-exclusion chromatography and ligand blotting, most PFOA was in protein-bound form in male and female rat plasma, and the primary PFOA binding protein in plasma was serum albumin. PFOA binding to rat serum albumin (RSA) in the gas phase was observed by electrospray ionization MS. 19F NMR experiments revealed that binding to RSA caused peak broadening and chemical shift changes of PFOA resonances, and on the basis of this observation, the dissociation constant was determined to be ∼0.3 mM. The dissociation constants for PFOA binding to RSA and human serum albumin (HSA) and the numbers of PFOA binding sites on RSA and HSA were also determined by a separation method using microdesalting columns. No significant difference was found between PFOA binding to RSA and PFOA binding to HSA. The dissociation constants for binding of PFOA to RSA or HSA and the numbers of PFOA binding sites were in the range of 0.3-0.4 mM and 6-9, respectively. On the basis of these binding parameters and the estimated plasma concentration of serum albumin, greater than 90% of PFOA would be bound to serum albumin in both rat and human blood.

Introduction Man-made organic fluorochemicals are used in industry as polymer additives, lubricants, fire retardants and suppressants, pesticides, and surfactants (1). Because of the physical and chemical properties of the carbon fluorine bond, many fluorochemicals are stable molecules that are resistant to biodegradation. The relative stability of these compounds has created focus on potential persistence in the environment. Furthermore, the detection of organic fluorine in human blood (2) has generated questions about the relationship between organofluorine blood levels and potential health effects. PFOA,1 a compound in which all of the aliphatic hydrogens of straight chain caprylic acid are replaced by fluorine, is a commercially important organic fluorochemical. Persistence of PFOA in the human body has been suggested (3), and the level of PFOA in the blood of workers at a PFOA manufacturing facility has been measured (4). Extensive studies of the ADME of PFOA in rats demonstrated that PFOA was distributed primarily to the plasma, liver, and kidney and was not metabolized. Urinary elimination of PFOA, the major excretion route of PFOA in rats, was significantly slower in male rats than in female rats, resulting in a much longer half-life of PFOA in male rats (15 days as compared to less than 1 day in female rats) (5-8). The * To whom correspondence should be addressed. Tel: 302-366-5214. Fax: 302-366-5003. E-mail: [email protected]. 1 Abbreviations: ADE, absorption, distribution, and elimination; ADME, absorption, distribution, metabolism, and elimination; COSY, correlation spectroscopy; ESI, electrospray ionization; FID, free induction decay; HSA, human serum albumin; PFOA, perfluorooctanoic acid; PVDF, polyvinylidene fluoride; RSA, rat serum albumin; SEC, sizeexclusion chromatography.

mechanism of sex-dependent elimination of PFOA in rats has not been well-understood. It has been suggested that female rats possess an active renal transport system for PFOA that is not expressed in male rats (5) or that PFOA binds to male rat specific proteins (9). PFOA is also considered a potent peroxisome proliferator (10-12), and its induction of liver peroxisome proliferation in male rats is far more pronounced than that in female rats (13, 14) presumably due to its slow elimination and accumulation in the livers of male rats. The ADE of PFOA in humans are not well-defined. However, it was reported that PFOA persists in the plasma of occupationally exposed workers, even though this persistence was not associated with any hepatic toxicity (15). It has long been suggested that PFOA is circulated throughout the body by noncovalently binding to plasma proteins (16). However, the detailed plasma protein binding properties of PFOA, which are potentially of great importance for understanding and predicting the ADE of PFOA in humans, have not been carefully investigated. This motivated us to compare of the protein binding states of PFOA in male and female rat plasma by SEC, to identify PFOA binding protein by ligand blotting, and to determine the binding parameters by ESI MS, 19F NMR, and microdesalting column separation.

Experimental Procedures Materials. RSA and HSA (∼99% and fatty acid free) were purchased from Sigma (St. Louis, MO). PFOA (∼96%, free acid) and 14C-PFOA (>99%, labeled at the carboxyl carbon, ammonium salt; activity, 2.11 GBq/mmol) were purchased from Aldrich (Milwaukee, WI) and Amersham Pharmacia Biotech (Piscataway, NJ), respectively. D2O (>99.9%) was obtained from Isotec (Miamisburg, OH). All other chemicals were from Sigma.

10.1021/tx034005w CCC: $25.00 © 2003 American Chemical Society Published on Web 05/16/2003

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Animals. Male and female Crl:CD(SD)IGS BR rats were obtained from Charles River Laboratories (Raleigh, NC). The Sprague-Dawley rat was chosen for this study because of the extensive experience with this strain and its suitability with respect to longevity, sensitivity, and low incidence of spontaneous diseases. At the time of dosing, rats were 6-8 weeks of age and the weight variation did not exceed 20% of the mean weight by dose group. Animals were housed individually in glass metabolism cages and provided with feed and water ad libitum. Animal rooms were targeted at a temperature of 23 ( 1 °C and a relative humidity of 40-60%. Animal rooms were artificially illuminated (fluorescent light) on a 12 h light/dark cycle. PFOA Administration. 14C-PFOA was administered by oral gavage at a dose of approximately 25 mg/kg. The test substance (specific activity, 0.158 MBq/mg) was dissolved in water and administered in a volume of 4 mL/kg body weight. The purity of the dosing solution, which was greater than 99.3%, was confirmed by HPLC. The chemical and radiochemical concentration were verified by liquid scintillation counting. Animals were fasted overnight prior to administration of the test substance. SEC. Plasma samples were collected from the tail vein 2 h after dosing rats with 14C-PFOA and were analyzed immediately after collection. Twenty microliters of plasma samples was applied to a Bio-Rad Bio-Sil SEC 125-5 column (300 mm × 7.8 mm) and eluted with PBS at a flow rate of 1 mL/min at room temperature. The absorption of eluate was monitored at 280 nm and collected in 0.5 mL fractions. Fractions were mixed with 7 mL of scintillation cocktail for determination of total radioactivity. Ligand Blotting. Unreduced protein samples were subjected to SDS-PAGE (4-20% gradient Ready Gel, Bio-Rad) and electrotransferred onto a PVDF membrane with a Tris/glycine buffer that did not contain methanol. The PVDF membrane was then incubated in buffer A (50 mM Tris-HCl and 20% glycerol, pH 7.4) at room temperature for 1 h before blotting. After it was washed three times with buffer B (10 mM Tris-HCl, 150 mM NaCl, pH 7.4) for 20 min each time, the membrane was incubated overnight at room temperature with 1.0 × 107 cpm of 14C-PFOA in buffer B. The membrane was then washed again three times with buffer B for 5 min each time, dried, and autoradiographed with a PhosphorImager (model 445SI, Molecular Dynamics). ESI MS. Protein ESI MS was performed on a Micromass Q-Tof II mass spectrometer, equipped with a Micromass Z spray ion source (Micromass, Manchester, U.K.). RSA with or without the addition of PFOA was dissolved in 50 µM ammonium bicarbonate, pH 7.0, and was infused into the mass spectrometer at a rate of 5 µL/min. The mass spectrometer was scanned from 800 to 6000 mass-to-charge ratios at a rate of 2-4 s per scan with the resulting positive ion spectra produced by averaging 10 min of accumulative data. The electrospray source conditions were as follows: cone voltage, 20-50 V; source temperature, 80 °C; desolvation temperature, 80-150 °C. 19F NMR. 19F NMR was performed on a 360 MHz Bruker AMX spectrometer (Bruker BioSpin, Bremmen, Germany) at 23 °C with a dual 1H/19F probe. Samples (600 µL in 5 mm NMR tubes) in PBS containing 0.05% NaN3 and 10% D2O were composed of RSA with a fixed concentration of either 12.5 or 25 µM and PFOA, whose concentrations were varied over a wide range. Spin-lattice relaxation times (T1) for resonances of PFOA in PBS were measured by the inversion-recovery technique and were found to be around 1 s. One-dimensional (1D) 90° pulse spectra were collected with 25 kHz sweep width, 5 s pulse delay, 16K data points, and 64-2048 scans. Two-dimensional (2D) COSY spectra were recorded with 512 complex t1 increments, 1024 t2 points, and 16 scans for each FID. Chemical shifts were referenced to the CF2 resonance of PFOA in PBS (see Results for resonance assignments), which is set at 0 ppm. The CF2 resonance showed the largest chemical shift changes as a result of protein binding of PFOA. Protein-ligand equilibrium binding constants were measured by taking advantage of the observation that once bound to

Han et al. proteins the chemical shift of certain resonance of the ligand changes and the extent of the change reflected the amount of the ligand bound to proteins. The theoretical and experimental procedures were described previously (17, 18). Briefly, if the fraction of ligand bound to protein was small, the dissociation constant Kd was given by the following equation:

[L]T )

n[P]T ‚ ∆δBapp - Kd ∆δ

(1)

where ∆δ is the net change in chemical shift of the resonance of the ligand (CF2 of PFOA in this case) due to the interaction of the ligand with the protein, [L]T is the total ligand concentration, [P]T is the total concentration of protein provided that the protein is monomeric under the experimental condition, n is the number of binding sites, and ∆δBapp is the apparent chemical shift change for the ligand in the bound state. Thus, a plot of [L]T vs 1/∆δ gives a straight line, which intercepts with the y-axis at -Kd. Microdesalting Column Separation. Micro Bio-Spin columns (Bio-Rad) were used to rapidly separate the proteinPFOA complex from free PFOA. These columns had a molecular mass cutoff of 6000 Da and were tested with bovine serum albumin, which showed (103 ( 6)% recovery rate of the protein. Quantification was accomplished by including 0.5 µM 14C-PFOA in each sample. Briefly, 50 or 60 µM of protein was titrated with 0.1-3 mM PFOA in PBS in a final volume of 70 µL. Samples were incubated at room temperature for 30 min before being loaded onto the preequalibrated columns. The columns were centrifuged at 1000g for 4 min. Free PFOA was retained on the column whereas protein-PFOA complex freely passed through the column. The radiochemical activity of 50-60 µL of the collected samples was determined, allowing quantitation of the protein-bound PFOA. The concentration of free PFOA, [L]F, was calculated accordingly. Dissociation constants of the binding, Kd, and the number of binding sites, n, were given by the following equation, which is a variant of Scatchard analysis (19):

[L]F [L]F Kd ) + r n n

(2)

where r is the moles of PFOA bound per mole of protein, [L]F is the concentration of unbound PFOA, and all other parameters stand for the same meaning as in eq 1. A plot of [L]F/r vs [L]F is a straight line with a slope of 1/n and an intercept of Kd/n.

Results PFOA Binding to Proteins in Rat Plasma as Determined by SEC. The protein binding state of PFOA in rat plasma was examined by a SEC method. Because the elimination of PFOA in rats is sex-dependent (5-8), plasma samples from 14C-PFOA-treated male rats and female rats were collected and analyzed. Figure 1 shows the size-exclusion chromatograms of plasma from male (A) and female (B) rats treated with 14C-PFOA, where elution of plasma proteins was measured at 280 nm (dotted lines) and the elution profile of 14C-PFOA was represented by the radioactivity (solid lines). The retention time of 14C-PFOA in PBS was around 20 min. Therefore, the coelution of 14C-PFOA with plasma proteins that was shown in Figure 1A,B demonstrates that in rat plasma, most, if not all, PFOA was associated with proteins. This finding is consistent with earlier reports (16), and it also shows that the extent of binding of PFOA to plasma proteins does not appear to be sex-dependent. Identification of PFOA Binding Protein in Rat Plasma by Ligand Blotting. Ligand blot assay has been widely and successfully used to identify specific

PFOA Protein Binding

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Figure 3. Identification of RSA-PFOA binding by ESI MS. Mass spectra of 25 µM fatty acid free RSA with (A) or without (B) the addition of 200 µM PFOA in 50 µM ammonium bicarbonate, pH 7.0. The charge states of RSA or RSA-PFOA complex and the numbers of PFOA molecules in association with RSA are indicated. Figure 1. Size-exclusion chromatograms of plasma from 14CPFOA-treated male (A) and female (B) rats. Both the UV absorption at 280 nm (dotted lines) and the radiochromatograms (solid lines) are shown. Plasma samples were applied to a BioRad Bio-Sil SEC 125-5 column (300 mm × 7.8 mm) and eluted with PBS buffer at a flow rate of 1 mL/min at room temperature. The retention time of standard 14C-PFOA dissolved in PBS on the same column was around 20 min and was marked as “Free PFOA” on the figure.

Figure 2. Identification of PFOA binding proteins by ligand blotting. A 2.5 µL amount of female (a and b) and male (c and d) rat plasma was run on a 4-20% gradient gel and stained with Coomassie blue (a and c), and another gel run in parallel was transferred to a PVDF membrane, which was then blotted with buffer containing 1 × 107 cpm of 14C-PFOA (b and d).

binding proteins (20-23). In the current study, male and female rat plasma proteins were fractionated on a 4-20% gradient gel. The gel images are shown in Figure 2c,a, respectively. After they were transferred onto a PVDF membrane, plasma proteins were incubated with Tris buffer containing 14C-PFOA. The resulting autoradiograms for both male and female rat plasma samples are shown in Figure 2d,b. Only one major protein at 66 kDa showed PFOA binding capability. On the basis of its molecular weight and its large quantity on the gel relative to other plasma proteins, this protein was considered to be RSA. Binding of PFOA to RSA Studied by ESI MS. Ligand blotting experiments demonstrated that RSA is the primary PFOA binding protein in rat plasma. To further characterize the PFOA-RSA complex, the binding of PFOA to RSA in a physiological buffer was investigated by using a Q-Tof II mass spectrometer equipped with an ESI interface. Figure 3 shows the mass

spectra of RSA with (A) and without (B) the presence of PFOA in 50 µM ammonium bicarbonate at pH 7.0. Only the mass-to-charge (m/z) ratios between 3260 and 4100 are shown in Figure 3 for the purpose of clarity. Under mild electrospray source conditions and a solution pH of 7.0, RSA ions are highly charged showing a charge state distribution from +17 to +20 (Figure 3B). These are the charge states of RSA while maintaining its native conformation. Because denatured RSA molecules expose more sites for protonation, they have lower m/z values on the mass spectrum (m/z 800-2000, data not shown). In principle, the binding of PFOA to RSA should lead to an increase in mass by 414 Da for each bound PFOA molecule. After an 8-fold molar excess of PFOA was added to the protein solution, adduct ions of [RSA + mPFOA + iH]i+ (i ) 17-20; m ) 1-6) were observed in addition to the protein ions [RSA + iH] i+ (i ) 17-20) (Figure 3A; the peaks are marked with corresponding values of i and m). These data confirmed the binding of PFOA to rat albumin and further suggested that the binding is sustained in the gas phase under proper conditions. The stoichiometry of the PFOA-RSA complex in the gas phase determined by MS suggests that RSA may possess as many as six distinct binding sites for PFOA. Binding Affinity Determination by 19F NMR. PFOA was found to exhibit a significant fluorine chemical shift change upon binding to serum albumin. Figure 4A shows the chemical structure of PFOA and Figure 4B shows the 1D 19F NMR spectrum of PFOA in PBS buffer. For convenience, the resonance that exhibited the largest chemical shift change due to protein binding was referenced as 0 ppm. Figure 4C shows the 1D PFOA spectrum in the region between -10 and 0 ppm and the corresponding 2D COSY spectrum. The J coupling connections between CηF3 and CF2 and between CβF2 and CδF2 were shown with dotted and dashed lines, respectively. The resonance of CηF3 was assigned by integration, and CβF2 was assigned by comparing with the 1D 19F NMR spectra of shorter chain length of perfluorinated fatty acids (C7 to C3, data not shown). The final assignments were labeled on the 1D spectrum of PFOA in Figure 4. Figure 5A shows the peak broadening and downfield shift of the CF2 resonance of PFOA (200 µM) in the presence of 100 µM RSA at pH 7.4. Binding isotherms

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Figure 5. Determination of PFOA-RSA binding affinity by 19F NMR. (A) 19F NMR spectra (arbitrary y-scale) showing C F  2 resonance of PFOA (200 µM) in PBS solution (top) and in the presence of 100 µM of fatty acid free RSA (bottom). (B) Binding isotherms were obtained at 23 °C and plotted according to eq 1 (Materials and Methods). Protein concentrations ([P]T) and dissociation constants (Kd) are indicated. Figure 4. Resonance assignment of PFOA 19F NMR spectrum. (A) Chemical structure of PFOA. (B) One-dimensional 19F NMR spectrum of 1 mM PFOA in PBS, pH 7.4. The resonance that exhibited the largest chemical shift change upon binding to albumin was referenced as 0 ppm. (C) The -10 to 0 ppm region of the 1D and 2D COSY PFOA spectra. Assignment based on the J coupling was shown on the COSY spectrum. J coupling connections between CηF3 and CF2 and between CβF2 and CδF2 were labeled by dotted and dashed lines, respectively.

were obtained by titrating RSA with PFOA (Figure 5B). Control experiments (data not shown) indicated that in the absence of protein, the PFOA resonances did not shift nor broaden even at the highest concentration that was used in the titration experiments. On the basis of eq 1 (Materials and Methods), the interception of the linear binding isotherm with the y-axis is equal to -Kd and is independent of the protein concentration. Figure 5B shows two binding isotherms representing PFOA binding to 12.5 and 25 µM RSA in PBS, respectively. The two isotherms possess different slopes but yield comparable Kd values of 0.31 ( 0.15 and 0.27 ( 0.05 mM, respectively. Here, errors associated with Kd are from the scatter of the NMR measurements. Their average (0.29 ( 0.10 mM), therefore, is obtained to represent the Kd value that is determined by NMR (see Table 1). Quantitative Comparison of PFOA Binding to RSA and HSA by Microdesalting Columns. The 19F

Table 1. Dissociation Constants (Kd) of Binding between PFOA and RSAs and HSAs and the Number of PFOA Binding Sites (n) on RSAs and HSAs Kd (mM)

NMRa

n

micro-SECb micro-SECb

RSA

HSA

0.29 ( 0.36 ( 0.08c 7.8 ( 1.5

0.38 ( 0.04 7.2 ( 1.3

0.10c

a Average of the two K values (0.31 ( 0.15 and 0.27 ( 0.05 d mM) obtained by NMR. b Values were obtained from three independent experiments, and their standard deviations are shown. c On the basis of the result of unpaired t-test at 95% confidence interval, the difference of Kd values determined by NMR and micro-SEC is statistically insignificant.

NMR study was used to determine the affinity of PFOA binding to RSA. However, to estimate the PFOA binding capacity of serum albumin, it is also necessary to know the total number of PFOA binding sites on albumin. Microdesalting columns were used to separate and quantify protein-bound and unbound PFOA. Scatchard plots were prepared to illustrate the binding between PFOA and serum albumin. Ultrafiltration, the conventional method for fast separation of protein-ligand complex from free ligand, failed because PFOA completely nonspecifically bound to the membrane of the ultrafiltration unit (Amicon Ultrafree centrifugal filter with Biomax membranes, data not shown). The microdesalting columns were determined to be suitable for this

PFOA Protein Binding

Figure 6. Determination of binding affinity and number of binding sites by microdesalting column separation. Binding isotherms plotted according to eq 2 (Materials and Methods) were obtained from PFOA binding to 50 µM fatty acid free RSA and 50 µM fatty acid free HSA in PBS at room temperature.

study because in control experiments serum albumin was nearly 100% recovered after centrifugation of the column and free PFOA in PBS was more than 98% retained on the column (14C-PFOA was used as marker, data not shown). Using this technique, the binding of PFOA to RSA and HSA was compared and the data were represented by Scatchard plots (Figure 6). The resulting binding parameters are summarized in Table 1. The dissociation constants of PFOA binding to RSA and HSA were 0.36 ( 0.08 and 0.38 ( 0.04 mM, respectively. The numbers of binding sites of PFOA on RSA and HSA were also determined, which are in a range of 6-9 for both RSA and HSA (Table 1). This is in good agreement with the value determined by ESI MS. These data indicate that binding of PFOA to serum albumin is quantitatively similar between rats and humans.

Discussion A variety of techniques were used in this study to elucidate the protein binding properties of PFOA in rat and human plasma. Most notable are the ESI MS and 19F NMR methods. ESI MS is an emerging and fast-developing technique in the study of noncovalent protein-protein, proteinDNA, and protein-ligand complexes (24-26). In contrast to most established methods, MS detects the formation of noncovalent protein complexes in the gas phase. This is made possible by using extremely “soft” ionization parameters in the ion source. Although the validity of the gas phase protein complex remains controversial (27), there are an increasing number of examples, which

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demonstrate that if used with caution, ESI MS is an efficient and unique technique for identifying noncovalent protein complexes (28-32). Moreover, because it is capable of observing discrete combinations of ligand and protein in a complex by measurement of the molecular masses of all forms in the complex, ESI MS is by far the most straightforward way to obtain the stoichiometry of a complex. In our study, we were able to determine the stoichiometry of PFOA-RSA binding (at least 6:1) on a Q-Tof (Figure 3). This result is in agreement with the stoichiometry values that were determined in a solution phase by a column separation method (Table 1), which strongly supports the validity of the ESI MS method in determination of protein-ligand binding. NMR methodology has been successfully used in the study of protein-ligand binding affinity (17, 18). Binding to proteins caused the changes of the ligands’ (PFOAs, in this case) chemical shift and bandwidth, and these effects allowed the determination of the binding isotherms by ligand titration experiments. The 19F NMR spectrum of PFOA has been reported and assigned previously by Goecke and co-workers. (33). However, the COSY spectrum of PFOA was not shown in this paper, which makes justification of their spectral assignments impossible. The resonance assignments of the 1D PFOA 19F NMR spectrum presented here (Figure 4) confirmed Goecke et al.’s assignment on CηF3 but did not agree with any of the six remaining assignments. The 2D COSY spectrum of PFOA is presented in Figure 4C to show the J coupling connectivity, which is the basis for our NMR peak assignments. Plasma Protein Binding and Plasma Persistence of PFOA. The binding of small molecules, such as industrial chemicals and drugs, to plasma proteins has long been recognized as one of the major determinants of their disposition in the body (34). It is known that PFOA has a much longer plasma half-life in male rats than in female rats. To examine if protein binding is a factor in the persistence of PFOA in male rat plasma, we compared the extent of plasma protein binding of PFOA in male rats to that of female rats. SEC and ligand blotting methods revealed that most of the PFOA was bound to serum albumin regardless of gender (Figures 1 and 2), suggesting that there is no apparent correlation between PFOA persistence and PFOA serum albumin binding in plasma. Serum albumin in plasma has a large capacity for binding PFOA (6-9 binding sites per molecule and mM concentration in plasma), giving an estimated free fraction of less than 5% based on the SEC data (not shown). The fact that female rats (but not male rats) can still quickly eliminate PFOA suggests that the role of RSA binding in the distribution and elimination of PFOA could be much more complex. It is still controversial as to what roles serum albumin plays in organic anion transport in tissues. There are examples demonstrated that at physiological albumin concentration, the rate of hepatocyte ligand uptake was proportional to the concentration of the unbound ligands, which suggests that it was the unbound ligands that drove ligand uptake (35). However, in other cases, the uptake rate was enhanced greatly by ligand binding to serum albumins. This kinetic behavior has been observed in vivo (36) and in the isolated perfused liver (37-39) and heart (40), hepatocyte suspensions (41-44), and cultures (45, 46). Fewer studies have focused on the role of serum albumin during renal secretion of organic anions, although avail-

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able evidence also supports an albumin-stimulated transportation mechanism. It has been shown that serum albumin-facilitated renal tubular secretion of p-aminohippurate and methotrexate and this stimulatory effect are not affected by the different binding affinities of these two ligands to albumin (47). Therefore, binding to serum albumin alone will not explain the persistence of PFOA in rats and other mechanisms should be explored. The ligand blotting method was used to identify PFOA binding protein in plasma (Figure 2). However, it is worth noting that this method will not do well in the identification of low abundance protein(s). Therefore, we cannot completely rule out the possibility of the existence of protein(s) with low abundance in plasma but high affinity to PFOA. Further investigation on this issue might be valuable to our understanding on the persistence of trace amounts of PFOA in human blood where high PFOA binding capacity (such as albumin) is not necessarily required. Serum Albumin’s PFOA Binding Properties. Serum albumin, the most abundant protein in plasma, is present in the blood at ∼0.6 mM. It is the major transport protein for free fatty acids but is also capable of binding an extraordinarily diverse range of steroid hormones, metabolites, drugs, and other organic compounds. It has been proposed that there are five binding sites on HSA for medium or long chain fatty acids (48-50) and the binding affinity is relatively high (Kd range from 0.05 to 1.0 µM (51)). In contrast, HSA possesses more PFOA binding sites (n ) 6-9), but the binding affinity is lower (Kd ) 0.38 mM, Table 1). Because the critical micelle concentrations of PFOA free acid and its salts are high (∼9 and ∼30 mM, respectively) (52), PFOA is believed to be monomeric, i.e., not self-associated by forming micelles under the conditions that the binding parameters were measured. The nearly perfect linearity of the binding isotherms shown in Figures 5 and 6 suggests that PFOA-serum albumin binding is noncooperative. It is not known if PFOA shares the same binding sites on serum albumin with fatty acid. However, if this was the case, competition for binding sites would be much more favorable to fatty acids. It is estimated, based on the Kd values and apparent number of PFOA binding sites, that greater than 90% of PFOA would be bound to serum albumin in blood provided that the PFOA concentration in blood is far less than the value of Kd and that most of serum albumin PFOA binding sites are available. In our study, we also compared PFOA binding properties between RSA and HSA. Because serum albumin is the major PFOA binding protein in plasma, comparison of the PFOA binding properties of serum albumins from different species is important for understanding speciesrelated blood kinetics and toxicity of PFOA and will be the basis for extrapolation of the animal models of PFOA blood distribution and elimination to humans. Our results demonstrated that RSA and HSA possess similar, if not identical, PFOA binding properties (Table 1).

Acknowledgment. We thank the Association of Plastics Manufacturers in Europe (APME) for sponsoring this study. We are very grateful to Drs. Matthew S. Bogdanffy and Paul M. Hinderliter for many helpful and stimulating discussions. We also thank B. Robinson and S. Carpenter for excellent in-life technical assistance.

Han et al.

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