Faculty of Pharmacy and Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
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ABSTRACT |
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Determination of the BSA-palmitate high-affinity
binding constant
(Ka)
traditionally relied on the heptane-water partitioning technique. We
used this technique to calculate
Ka for the
BSA-[3H]palmitate
complex, to determine if
Ka was
independent of protein concentration, and to determine if the unbound
[3H]palmitate
concentration is constant at different BSA concentrations using
constant BSA-to-palmitate molar ratios (range 1:1 to 1:4). After
extensive extraction of non-[3H]palmitate radiolabeled
substances, the heptane-to-buffer partition ratio, in the absence of
BSA, was 702 ± 19 (mean ± SD,
n = 6). This value was much lower than
the predicted value of 1,376 and was highly dependent on which phase
(organic or aqueous) initially contained the
[3H]palmitic acid. The
data were consistent with the notion of self-association of
[3H]palmitate in the
aqueous phase. Ka
for the
BSA-[3H]palmitate
complex was determined to be similar (2.2 ± 0.1) × 108
M1 (mean ± SD,
P > 0.05) at all BSA concentrations
studied. At each BSA-to-palmitate molar ratio, the equilibrium unbound
ligand concentration was constant only at low BSA concentrations (<10
µM) and at low BSA-to-palmitate molar ratios (i.e., 1:1 and 1:2). At
higher BSA concentrations and molar ratios, the unbound ligand
concentration increased with an increase in protein concentration.
Hepatocyte uptake using the manufacturer-supplied radiolabeled product
was significantly higher than with the purified product, suggesting that a
non-[3H]palmitate
radiolabel is also a substrate for the uptake process.
heptane; partitioning; long-chain fatty acids; protein binding
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INTRODUCTION |
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IN PLASMA, NONESTERIFIED long-chain fatty acids are highly bound to circulating BSA. Because of protein binding, the unbound fraction is extremely small. Despite this, the hepatic extraction fraction is very large. Elucidating the mechanism for the extremely efficient hepatic removal of long-chain fatty acids has been the subject of much research work over the past 30 years.
The hepatic uptake process is typically studied in the presence of binding proteins, such as BSA, and tracer ligand concentration. BSA concentrations normally range from low (2 µM) to physiological (23). Use of tracer ligand concentration ensures that binding occurs to the high-affinity site of BSA. This allows investigators to calculate an apparent high-affinity equilibrium binding constant (Ka) for that site. Other investigators (29) choose to conduct uptake studies using constant BSA-to-ligand molar ratios (1:1, 2:1, 3:1), again encompassing a range of BSA concentrations. For all studies, the precise determination of the unbound fraction is critical in elucidating the uptake process.
Calculating an unbound fraction requires knowledge of the
Ka value for the
BSA-ligand complex. There are several methods currently available for
determining the unbound long-chain fatty acid concentration in the
presence of binding proteins. Some methods use spectrophotometric titration, utilizing fluorescent probes such as acrylodan-derivatized intestinal fatty acid binding protein (26) and doxorubicin-BSA (14). These probes undergo a wavelength shift when binding
fatty acids and as such are easy to analyze. Another method involves equilibrium dialysis. Protein-bound ligands are separated from the
unbound ligands by a physical barrier [e.g., polyethylene sheeting (9, 21)]. A drawback of this method is that it cannot be
used for determining the unbound fatty acid fraction in the presence of
high BSA concentrations (8). These methods result in estimates of
Ka that ranged
from a low of 0.3 × 108
M1 (30) to a high of 4.6 × 108
M
1 (11, 24). Use of the
lower estimates leads one to calculate higher unbound fractions,
whereas using the higher
Ka values leads to much lower calculated unbound fractions.
The simplest and most widely used technique employs partitioning of
long-chain fatty acids between an organic and an aqueous phase.
However, this technique results in
Ka values from
0.2 × 108
M1 (15) to 1.4 × 108
M
1 (6). The order of
magnitude difference in
Ka values makes interpretation of uptake data difficult. Moreover, this technique requires calibration. We have previously described (10) potential problems that occur in calibrating this method due to radiolabeled impurities. In this report, we repeat and extend those observations to
determine the effect of both hydrophilic and lipophilic radiolabeled impurities on the Ka for the
BSA-palmitate complex. We use this method to test the hypothesis that
the BSA-palmitate
Ka value is
constant at all BSA concentrations. It has been suggested that Ka for the
BSA-long-chain fatty acid complex may not be a constant but may be
dependent on BSA concentration (12). We extend our studies to determine
the unbound palmitate concentration at different BSA concentrations
when the protein-to-ligand molar ratio is kept constant.
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MATERIALS AND METHODS |
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[9,10-3H]palmitic acid (56.5 Ci/mmol) with a radiochemical purity of 97.8% was obtained from NEN. BSA (essentially fatty acid-free) was obtained from Sigma Chemical (St. Louis, MO). All other chemicals were obtained from Sigma Chemical with the exception of heptane, which was supplied by Fisher Scientific (Pittsburgh, PA). The aqueous buffer used throughout all experiments was PBS, which had a composition of (in mM) 137 NaCl, 2.68 KCl, 1.65 KH2PO4, 8.92 Na2HPO4, and 3 NaN3, with pH adjusted to 7.4 using 0.1 N NaOH.
Construction of Incubation Vessel
Glass scintillation vials (20-ml borosilicate glass with white urea screw caps; Fisher), 150-ml Erlenmeyer flasks, and 500- and 1,000-ml glass bottles (Wheaton) with black screw caps were siliconized by immersion in 2% toluene solution of dimethyldichlorosilane for 1 h, air-dried, rinsed thoroughly with distilled water, and dried before use (for the 150-ml Erlenmeyer flasks, we used a plastic stopper instead of screw caps). A sampling tube (1.6-mm ID capillary tubes; Corning) was inserted through a predrilled hole in the cap of each vessel. The capillary tube was secured to the cap using Press-Tite contact cement (no. 6; LePage, Ontario, Canada). A Sure-cap (Baxter Scientific Products) was used to cover the outer end of each capillary tube.Purification of [3H]Palmitic Acid
[3H]palmitic acid was purified as previously described (4, 10). A 1-ml sample of the manufacturer-supplied solution of [3H]palmitic acid was added to 0.98 ml research quality distilled water (18 MHeptane-Buffer Partitioning of [3H]Palmitic Acid and Measurement of Radioactivity
The partitioning of [3H]palmitate between heptane and buffer was determined as described previously (10, 16, 30). Vessels were incubated at 37°C in an oscillating (120 rpm) water bath (model G760; New Brunswick Scientific) for up to 120 h. The buffer phase was sampled by inserting a Hamilton syringe (100 µl) through the capillary tube. The contents of the syringe were added directly to a scintillation vial. The heptane phase was directly sampled using an Eppendorf pipette. The outsides of the pipette tips were wiped with a tissue prewetted with heptane. Both the pipette tip and its contents were added to a scintillation vial for determination of radioactivity. Radioactivity was determined using an LS6500 liquid scintillation counter (Beckman Instruments) with automatic quench correction after the addition of Ready Safe (Beckman) scintillate.The following six protocols were followed to determine the effect of radiolabeled impurities on the [3H]palmitate heptane-to-buffer partition ratio in the absence and presence of BSA.
Protocol 1. Experiments were conducted to investigate the effect of incubation time on the partitioning of [3H]palmitic acid between heptane and buffer in the absence of BSA.
Protocol 2. Experiments were conducted to investigate the effect of organic and aqueous phase volumes on the [3H]palmitate heptane-to-buffer partition ratio. The heptane-to-buffer volume ratio ranged from 1 to 0.005.
Protocol 3.
Experiments were conducted to investigate the effect of the initial
addition of
[3H]palmitic acid
(tracer concentration 2 nM) to the heptane phase, the subsequent
evaporation of the heptane, and the addition of fresh heptane to the
aqueous phase on the partition ratio value. Adding the
[3H]palmitic acid to
the heptane phase with subsequent evaporation of the heptane prevented
the initial high concentration that would be expected if the
[3H]palmitic acid was
added directly to the aqueous phase, thus reducing the chance of
aggregate formation. Adding the
[3H]palmitic acid
directly to the aqueous phase may be expected to be associated with the
lowest heptane-to-buffer partition ratio by virtue of the fact that the
high [3H]palmitic acid
concentration in the aqueous phase may form aggregates.
Protocol 4. Experiments were conducted to determine the partition ratio when hydrophilic radiolabeled impurities were minimized through a series of extractions. After equilibration of [3H]palmitate, the heptane phase was harvested and layered over fresh buffer. This maneuver was repeated several times in an attempt to reduce the hydrophilic radiolabeled impurities. It may be expected that this set of experiments represented the highest partition ratio (owing to the minimal hydrophilic radiolabeled impurities).
Protocol 5. Experiments were designed to investigate the heptane-to-buffer partition ratio in the presence of BSA (concentration range 0.1-800 µM). A series of extraction steps was followed that reduced the contribution of both hydrophilic and lipophilic radiolabeled impurities to the partition ratio values (PR+). Table 1 outlines the initial conditions and successive extraction procedure. The initial condition shows the volume of heptane and buffer phase used. Table 1 indicates the volume of heptane and buffer phase that, after equilibration, was sampled, discarded, and replaced with the same volume of fresh heptane and/or buffer for extraction 1. Again, after equilibration, a second extraction procedure was performed. The extraction procedures were repeated six times. In each case, the [3H]palmitic acid was initially added to the aqueous phase.
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Protocol 6. The unbound [3H]palmitate fraction was determined at various BSA concentrations, while the BSA-to-palmitate molar ratio was kept constant.
Hepatocyte Isolation
Studies were carried out in accordance with the University of Manitoba Animal Care Committee. Hepatocytes were isolated from female Sprague-Dawley rats (body wt, 125-150 g), as previously described (6). Rats were housed in a temperature- and light-controlled room (22°C; 12:12-h light-dark cycle starting at 0600) and allowed Prolab animal diet (Agway County Foods) and water ad libitum. Livers were perfused in situ at 20 ml/min initially with oxygenated Swim's S-77 medium containing 5 mM EDTA and finally with Swim's S-77 medium containing 25 mg/dl collagenase and 5 mM CaCl2 after an intraperitoneal injection of pentobarbital (50 mg/kg). Perfused livers were excised, combed free of connective tissue, and filtered through 50 mesh followed by 200 mesh stainless steel filters (Sigma Chemical). After three to four centrifugation steps (each at 300 rpm) to eliminate any nonviable cells, the viable isolated cells were stored at room temperature and used within 2 h of isolation. Immediately before experimentation, cells were equilibrated to 37°C. Trypan blue exclusion was typically >90% before and after the uptake studies.Uptake Procedure
Hepatocytes were added to solutions of PBS containing fatty acid-free BSA and tracer concentrations of either the purified [3H]palmitic acid or the manufacturer-supplied [3H]palmitic acid. At specified times, 1.0-ml aliquots of cell suspension were sampled, immediately filtered through GF/C glass microfiber filters (Fisher Scientific) by vacuum, and washed with 5 ml ice-cold PBS to stop uptake and to remove adherent extracellular fluid. Cell-associated radioactivity was assessed by liquid scintillation counting (Beckman LS6500TA) using Ready Safe scintillate (Beckman).Data Analysis
The heptane-to-buffer partition ratio was calculated as the total radioactivity in the heptane phase divided by the total radioactivity in the buffer phase. The predicted [3H]palmitic acid heptane-to-buffer partition ratio in the absence of a protein (PR
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(1) |
The unbound
[3H]palmitic acid
fraction () was calculated using
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(2) |
The apparent equilibrium binding constant (Ka) was calculated using
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(3) |
Hepatocyte [3H]palmitate space was calculated from the ratio of the recovered cell-associated radioactivity to the concentration of radioactivity in the bathing medium. The [3H]palmitate clearance rate was calculated from the least-squares linear regression coefficient for the slope of the plot [3H]palmitate space divided by the uptake interval.
Data were analyzed with ANOVA and Tukey's multiple comparison test (3) and are reported as means ± SD, unless otherwise specified.
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RESULTS |
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[3H]Palmitic Acid Partition Ratio in the Absence of BSA
The time required to reach equilibrium for the partitioning of [3H]palmitic acid between 3.5 ml heptane and 700 ml buffer (volume ratio 0.005) is shown in Fig. 1. When the [3H]palmitic acid was initially added to the heptane phase, the partition ratio value at 24 h (878 ± 46; n = 6) was significantly greater (P < 0.01) than the 48-h value (695 ± 40; n = 6), which was not statistically different from the values at 72, 96, or 120 h. Organic and aqueous phases were therefore sampled at 48 h in all subsequent experiments that used a 0.005 heptane-to-buffer volume ratio. For smaller volume ratios, samples were obtained after 24 h, since our previous work has shown that equilibrium was achieved within 24 h (10).
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With the addition of [3H]palmitic acid to the heptane phase and the subsequent evaporation of the heptane (allowing the [3H]palmitic acid to be slowly incorporated into the aqueous phase), the heptane-to-buffer partition ratio at 24 h was much lower (259 ± 88; n = 3) than when the [3H]palmitic acid was initially added to the heptane phase without heptane evaporation (878 ± 46; n = 6). Equilibrium was reached after 48 h of incubation (545 ± 49; n = 6). When the [3H]palmitic acid was added directly to the aqueous phase, the 24-h heptane-to-buffer partition ratio was only 44 ± 15 (n = 4). Equilibrium was reached after 96 h of incubation (237 ± 8; n = 11). The partition ratio value in this case was statistically lower than the other two values, suggesting that formation of dimers or some other [3H]palmitate aggregates may be responsible for the low partition ratio values (see Fig. 1).
Table 2 shows the dependence of the partition ratio on the heptane-to-buffer volume ratio. As the volume of the aqueous phase increased (decreasing volume ratio), the partition ratio increased. This is compatible with the notion of hydrophilic radiolabeled substances that become less important (diluted) as the volume of the aqueous phase increases (10).
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Because radiolabeled impurities greatly affect the equilibrium binding constant value (6), we attempted to further decrease the degree of contamination. To this end, we performed a series of extractions. In these experiments, the [3H]palmitic acid was initially added to the heptane phase. After 24 h of incubation, the heptane phase was harvested and added to fresh buffer (extraction 1). After the second 24-h incubation, the heptane phase was again harvested and added to fresh buffer (extraction 2). This maneuver was repeated until the observed [3H]palmitic acid partition ratio did not change after further extraction. The heptane-to-buffer volume ratio in this series of experiments was kept constant at 0.3. Figure 2 shows that the initial heptane-to-buffer partition ratio value was 61 ± 0.6 (n = 6) and increased to a maximum of 702 ± 19 (n = 6).
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[3H]Palmitic Acid Partition Ratio in the Presence of BSA
Figure 3 depicts the [3H]palmitic acid buffer-to-heptane partition ratio values after successive extractions of the radiolabeled impurities (see Table 1; note that Fig. 3 shows buffer/heptane, not heptane/buffer, values.) Initially (no extraction), we observed that the partition ratio was dependent on BSA concentration. The deviation from linearity in Fig. 3 became less apparent with successive extractions. After extraction 6, the [3H]palmitic acid buffer-to-heptane partition ratio was linearly related to BSA concentration. At 800 µM BSA there existed a slight but statistically insignificant deviation from linearity. Table 3 shows that Ka, calculated by Eq. 3 and using our experimentally determined PR
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Table 4 shows the [3H]palmitic acid concentration at various BSA concentrations, using different BSA-to-ligand molar ratios. In all cases, the unbound ligand fraction decreased with an increase in the BSA concentration. The unbound [3H]palmitic acid concentration was not statistically different at low BSA concentrations (i.e., <10 µM) when the BSA-to-ligand molar ratio was 1:1 and 1:2. At higher BSA-to-ligand molar ratios (i.e., 3:1 and 4:1) the unbound ligand concentration increased with an increase in BSA concentration. In all cases, at BSA concentrations between 40 and 800 µM, the unbound ligand concentration increased with an increase in BSA concentration.
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Hepatocyte Clearance Rates
[3H]palmitate hepatocyte uptake curves in the presence of 5, 50, and 500 µM BSA are shown in Fig. 4. The apparent intrinsic clearance rates for the slopes of these plots using purifed [3H]palmitate were statistically lower than those using the manufacturer-supplied radiolabel. The total [3H]palmitate clearance rates (in means ± SE) using purified [3H]palmitate were (5.9 ± 0.4) × 10
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DISCUSSION |
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Heptane-buffer partitioning of long-chain fatty acids has been widely
used for determining the unbound fraction of long-chain fatty acids in
the presence of binding proteins. This technique is particularly well
suited for use at physiological BSA concentrations owing to the
increased solubility of palmitate in the heptane phase. However,
because there is a difference in the solubility of long-chain fatty
acids between the organic and heptane phases, this technique requires
calibration. We have recently evaluated this method (10) and reported
that calibration is difficult, making the technique unreliable unless
precautions are taken. The presence of hydrophilic and lipophilic
radiolabeled impurities makes estimation of the partition ratio, in the
absence of binding proteins, difficult. Partition ratio values may vary
by an order of magnitude and, indeed, this is what has been reported in
the literature (10, 15, 16, 27, 28). Lower heptane-to-buffer partition
ratio (PR) values were
reported by Smith and Tanford (28) and Fleischer et al. (15) of ~100,
while higher values (~1,400) were reported by Simpson et al. (27).
Although some of the problems associated with radiolabeled impurities
may be overcome, the presence of low-level lipophilic impurities makes
this technique unreliable for use with higher BSA concentrations, i.e.,
>100 µM (6). Hence, it is difficult to test the hypothesis that
Ka for the BSA-long-chain fatty acid complex is constant (12).
In the present study we confirmed our earlier report (10) showing the
[3H]palmitic acid
heptane-to-buffer partition ratio dependence on heptane-to-buffer
volume ratio. This dependence suggested the presence of radiolabeled
impurities. Our experimentally determined PR maximum value was much
lower (702 ± 19) than the predicted maximum of 1,376, despite
efforts to minimize the hydrophilic radiolabeled substances by using a
small heptane-to-buffer volume ratio (0.005). Thus radiolabeled
impurities and/or
[3H]palmitate
aggregates must be present despite our purification and dilution
efforts. Previous work (10) using gas chromatography/mass spectrometry
analysis showed the presence of glycerol monopalmitate and glycerol
monostearate as major lipophilic radiolabels. However, these monoglycerides seem unlikely to be responsible for the
differences observed in Fig. 1. Hydrophilic radiolabeled substances
could not be analyzed with gas chromatography/mass spectrometry because they were below detection limits. Because the partition ratio values
were dependent on which phase initially contained the radiolabel (Fig.
1), the possibility of
[3H]palmitic acid
aggregates is likely. These aggregates may function as the initial
phase of micelle formation. The presence of long-chain fatty acid
dimers has been postulated by Goodman (16) and a dimerization constant
has been calculated by Mukerjee (22) to explain the low partition ratio
values. It is unlikely that micelle formation offers an alternative
explanation for our low partition ratio results, since the unbound
long-chain fatty acid concentration in the aqueous phase (<1 nM) was
much lower than the critical micellar concentraton.
In the presence of binding proteins radiolabeled impurities greatly affect the PR+ value, because small amounts of impurity can become large relative to unbound [3H]palmitate. At low BSA concentrations (i.e., <5 µM) a large portion of the [3H]palmitate is present in the heptane phase. Hydrophilic radiolabeled substances may significantly affect the resulting PR+ value, whereas lipophilic radiolabeled impurities do not affect the PR+ value. At higher protein concentrations, the reverse occurs. Much more of the [3H]palmitate is present in the aqueous phase by virtue of its binding to BSA. Hydrophilic radiolabeled impurities are not expected to affect PR+, but lipophilic radiolabeled impurities may significantly affect PR+ values because a much smaller amount of [3H]palmitic acid is present in the organic phase. Thus, in the case of high concentrations of binding protein, the PR+ values could be artificially high.
Our method for removing hydrophilic and lipophilic radiolabeled
impurities (Table 1) resulted in buffer-to-heptane partition ratios
that were linearly related to BSA concentration (Fig. 3), suggesting
that Ka is indeed
a constant. At 800 µM BSA there was a slight
(statistically insignificant) deviation from linearity. (The procedure
outlined in Table 1, i.e., the selected heptane and buffer volumes and
the volumes removed for each BSA concentration, is not steadfast; any
suitable heptane-to-buffer volume ratio will help reduce the
contribution of radiolabeled impurities.) We calculated
Ka from our data
using the equation
Ka = (1
)/(
Ca). The free fraction (
) was calculated using our experimentally determined PR+ value (from Fig. 3)
and PR
of 702. Using
PR
= 702 to calculate
rather than the expected value (1,376) assumed that all radiolabeled
constituents are candidates for protein binding. This assumption may be
valid, since reports have shown that when two carbon-18 fatty acids are
covalently bound in the middle (thus having free carboxyl groups) they
act as a single-chain fatty acid (monomer) but with lower protein
binding properties (13, 17). Thus, if
[3H]palmitic acid
aggregation occurs, it also may behave as a monomer and bind to BSA.
The present study gives important experimental evidence showing that
the equilibrium binding constant for the BSA-palmitate complex is
independent of protein concentration. Previous work designed to
investigate the role of binding proteins on the drug uptake process has
determined the apparent
Ka using low
protein concentrations and assumed it to be the same at higher protein concentrations (6, 23, 29). To date, no study has
critically assessed this assumption for long-chain fatty acids. Through
reducing the radiolabeled contaminants in the manufacturer-supplied
[3H]palmitate, we
offer the experimental evidence that supports this assumption.
Moreover, the average equilibrium binding constant value obtained at
all BSA concentrations
[Ka = (2.2 ± 0.1) × 108
M1; mean ± SE]
agrees with the values reported by Pond et al. (24) [(4.6 ± 0.3) × 108
M
1; mean ± SE]
and Bojesen and Bojesen (2) [1.0 × 108
M
1].
The unbound fractions in the latter studies were determined using
erythrocyte ghosts. Richieri et al. (26) reported
Ka values of 1.45 × 108
M
1 and 1.22 × 108
M
1 for the binding of
palmitate to human serum albumin and BSA, respectively, using
fluorescence emission of acrylodan-derivatized intestinal fatty acid
binding protein. Compared with other accepted methods,
heptane-to-buffer partitioning is the only method sensitive enough to
measure the unbound fatty acid concentration using high BSA (i.e.,
physiological) and tracer palmitate concentrations.
Previous reports suggest (29, 31) that the binding of long-chain fatty
acids to BSA, at a given BSA-to-ligand molar ratio, results in a
constant unbound ligand concentration over a range of BSA
concentrations. Our studies show (Table 4) that the unbound ligand
concentration is constant only at low BSA concentrations (10 µM)
and only at low BSA-to-palmitate molar ratios (i.e., 1:1 and 1:2). At
higher BSA concentrations and higher molar ratios, the unbound ligand
concentration increased with an increase in protein concentration. This
observation was reported by Spector et al. (31), using acidic buffer
solutions (pH 6.0). Our results are of particular importance to studies
using BSA-to-ligand constant molar ratios, which may require
reinterpretation.
We further investigated whether the radiolabeled impurities are extracted by hepatocytes. Figure 4 shows that uptake is greater for the unpurified [3H]palmitate than the purified ligand. Clearly, these impurities are substrates for the cellular uptake process. These substances become much more important at higher binding protein concentrations. As the BSA concentration was increased from 5 to 500 µM, the extent of uptake attributable to radiolabel impurities also increased. At 5, 50, and 500 µM BSA ~20%, 56%, and 84% of total uptake was attributable to the impurities, respectively. Thus studies designed to investigate the uptake process of highly lipophilic protein-bound ligands such as [3H]palmitic acid must purify the manufacturer-supplied radiolabel before use. An overestimate of the uptake rates may otherwise be obtained.
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ACKNOWLEDGEMENTS |
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This study was supported by a grant from the Medical Research Council of Canada (MT-13683). B. M. Elmadhoun is a recipient of the University of Manitoba Graduate Studentship.
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FOOTNOTES |
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Address for reprint requests: F. J. Burczynski, Faculty of Pharmacy, Rm. 410 Pharmacy Bldg., Univ. of Manitoba, 50 Sifton Road, Winnipeg, Mannitoba, Canada R3T 2N2.
Received 19 August 1997; accepted in final form 12 June 1998.
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