1 Department of Medicine and 2 School of Biological Sciences, University of Auckland, Auckland 1001, New Zealand
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ABSTRACT |
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Peptides purified by
HPLC are often in the form of a trifluoroacetate (TFA) salt, because
trifluoroacetic acid is used as a solvent in reversed-phase HPLC
separation. However, the potential effects of this contaminant in
culture systems have not been addressed previously. TFA
(108 to
10
7 M) reduced cell numbers
and thymidine incorporation into fetal rat osteoblast cultures after 24 h. Similar effects were found in cultures of articular chondrocytes and
neonatal mouse calvariae, indicating that the effect is not specific to
one cell type or to one species of origin. When the activities of the
TFA and hydrochloride salts of amylin, amylin-(1
8), and calcitonin
were compared in osteoblasts, cell proliferation was consistently less
with the TFA salts of these peptides, resulting in failure to detect a proliferative effect or wrongly attributing an antiproliferative effect. This finding is likely to be relevant to all studies of purified peptides in concentrations above
10
9 M in whatever cell or
tissue type. Such peptides should be converted to a hydrochloride or
biologically equivalent salt before assessment of their biological
effects is undertaken.
osteoblasts; organ culture; trifluoroacetic acid; peptides
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INTRODUCTION |
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AS A RESULT of rapid advances in the techniques of molecular biology, many new proteins are being identified, purified, and synthesized. Considerable effort is being expended in many areas of biology to determine the activities of these peptides, many of which play roles in cell regulation. Such studies are usually carried out in cell or tissue cultures with either native or synthetic peptides that have been purified by HPLC. Most proteins purified by reversed-phase HPLC contain trifluoroacetate (TFA salt), derived from trifluoroacetic acid (TFA), which is used as a protein-solubilizing and chaotropic agent in HPLC. Peptides purified in this way form salts with TFA, which binds specifically to free NH2 termini as well as to side chains of exposed lysine, histidine, and arginine residues.
One such peptide is amylin. Amylin consists of 37 amino acids and would
be predicted to contain four TFA ions for each peptide molecule. This
has been confirmed by direct measurement in one of the widely used
preparations that is commercially available (J. Salik, personal
communication, Bachem California, Torrance, CA). In vitro studies of
the actions of amylin have typically used peptide concentrations
between 1010 and
10
7 M, resulting in TFA
concentrations in the culture media of four times this level. The
potential effects of these concentrations of TFA on biological
processes in cells or tissues do not appear to have been considered
hitherto. Because TFA is known to have wide-ranging effects on
membranes and proteins, it is important that its potential impact as a
contaminant in in vitro cultures is understood. The present studies
arose from our investigations of the effects of amylin on bone cells,
but they address an issue that is potentially of great importance to
the assessment of the actions of any peptide purified by HPLC from
solutions containing chaotropic anions in any culture system.
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MATERIALS AND METHODS |
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Osteoblast-like cell culture. Osteoblasts were isolated by collagenase digestion of 20-day fetal rat calvariae. Calvariae were dissected aseptically, and the frontal and parietal bones were stripped of their periosteum. Only the central portions of the bones, free from suture tissue, were collected. The calvariae were treated twice with phosphate-buffered saline (PBS) containing 4 mM EDTA (pH 7.4) for 15 min at 37°C in a shaking water bath. After being washed once in PBS, the calvariae were treated twice with 3 ml of 1 mg/ml collagenase for 7 min at 37°C. After the supernatants from these two digestions were discarded, the calvariae were treated two more times with 3 ml of 2 mg/ml collagenase (30 min, 37°C). The supernatants of the latter two digestions were pooled and centrifuged, and the cells were washed in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum (FCS), suspended in further DMEM-10% FCS, and placed in 75-cm2 flasks. After 48 h, the media were changed to minimal essential medium (MEM) with 10% FCS. Confluence was reached within 5-6 days, at which time the cells were subcultured. After trypsinization with trypsin-EDTA (0.05%/0.53 mM), the cells were rinsed in MEM with 5% FCS and resuspended in fresh medium and then seeded at 5 × 104 cells/ml in 24-well plates (0.5 ml cell suspension/well, i.e., 2.5 × 104 cells/well). The cells were incubated under 5% CO2-95% air at 37°C. Ascorbic acid (50 mg/l) was added to the MEM used throughout. The osteoblast-like character of these cells has been established by demonstration of high levels of alkaline phosphatase activity and osteocalcin production (3) and a sensitive adenylyl cyclase response to parathyroid hormone and prostaglandin E2 (5).
Proliferation studies (cell counts and thymidine incorporation) were performed in subconfluent cell populations. Twenty-four hours after subculturing, cells were changed to serum-free medium with 0.1% bovine serum albumin for a further 24 h before the addition of the experimental compounds. Cell numbers were analyzed 24 h after addition of the peptide or vehicle by the detachment of cells from the wells by exposure to trypsin-EDTA (0.05%/0.53 mM) for ~5 min at 37°C. Counting was performed in a hemacytometer chamber. Results are expressed per well. [3H]thymidine incorporation was assessed by pulsing the cells with [3H]thymidine (0.5 µCi/well) 2 h before the end of the experimental incubation. [3H]phenylalanine incorporation was assessed by pulsing the cells with [3H]phenylalanine (1 µCi/well) for 4 h before the end of the experimental incubation. Experiments were terminated at 24 h by washing the cells in MEM, followed by the addition of 10% trichloroacetic acid. The precipitate was washed twice with ethanol-ether (3:1), and the wells were desiccated at room temperature. The residue was redissolved in 2 M KOH at 55°C for 30 min and neutralized with 1 M HCl, and an aliquot was counted for radioactivity. Results are expressed as disintegrations per minute per well. For cell counts and thymidine incorporation, each experiment was performed at least four times with experimental groups consisting of at least six wells.
Chondrocyte culture. Chondrocytes were isolated by the removal of cartilage slices from the articular surfaces of the knee joints of adult dogs under aseptic conditions. Slices were placed in DMEM containing 5% FCS (vol/vol) and antibiotics (penicillin 50 µg/l, streptomycin 50 µg/l, and neomycin 100 µg/l) and chopped finely with a scalpel blade. Tissue was removed and incubated at 37°C first with pronase (0.8% wt/vol for 90 min), followed by collagenase (0.1% wt/vol for 18 h) to complete the digestion. The cells were isolated from the digest by centrifugation (10 min at 1,300 rpm), resuspended in DMEM-5% FCS, passed through a nytex mesh screen of 90-µm pore size to remove any undigested fragments, and recentrifuged. The cells were washed and resuspended twice in the same media and seeded into a 75-cm2 flask containing DMEM-20% FCS. The cells were incubated under 5% CO2-95% air at 37°C until confluence. Chondrocytes were seeded into 24-well plates, as for osteoblasts.
Bone organ culture. Bone resorption studies were carried out in neonatal mouse calvariae as described previously (8). Hemicalvariae were preincubated for 24 h in Medium 199 with 0.1% bovine serum albumin and then changed to fresh medium containing peptide, TFA, or vehicle. Incubation was continued for a further 48 h. To assess DNA synthesis, [3H]thymidine (0.6 µCi/ml) was added in the last 4 h of the incubation, as described previously (6). There were 5-7 hemicalvariae in each group.
Conversion of peptides from the TFA salt to the hydrochloride salt. The hydrochloride salt of each peptide was produced by dissolving it in 3 mM hydrochloric acid (10 µmol peptide in 50 µl) and leaving it for 1 h at room temperature before freeze-drying (Savant Instruments, model SVC 100H, Holbrook, NY). This results in the substitution of hydrochloride for TFA salt, the latter being converted to TFA, which is volatile and is removed by lyophilization. Before use, the peptide was redissolved in pure water with sonication (Soniprep 150, West Sussex, England), cooled on ice for 15 s, and then stored at 4°C until required. The integrity of the peptides after this procedure was confirmed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (P2025A MALDITOFF, Hewlett-Packard).
Materials. Rat amylin, rat calcitonin,
and human calcitonin gene-related peptide were from Bachem California.
The rat amylin-(18) used in this study was a COOH-terminal amide
synthesized as described previously (2). TFA was obtained from BDH
Laboratory Supplies (Poole, Dorset, England). EDTA and collagenase were
obtained from Sigma (St. Louis, MO). Trypsin-EDTA, MEM, DMEM medium,
Medium 199, and FCS were from GIBCO (Grand Island, NY).
[3H]thymidine and
[3H]phenylalanine were
from Amersham International (Little Chalfont, Buckinghamshire, UK).
Statistical analysis. Data are presented as means ± SE. The significance of differences between groups was determined with Student's t-test. The comparisons to be made in each experiment were specified a priori, so no adjustment for multiple comparisons was necessary. Where several experiments have been shown in one figure, the data are expressed as the ratio of results in treatment groups to those in the control group and the P values shown were calculated with the data from the individual experiments before the data were pooled.
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RESULTS |
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The effects of TFA in concentrations of
109 to
10
7 M were studied in
cultured osteoblasts. There was a concentration-dependent reduction in
cell number, thymidine incorporation, and phenylalanine incorporation
at 24 h (Fig. 1). This suggests that both cell
proliferation and protein synthesis were impaired, although it should
be noted that the decrease in phenylalanine incorporation paralleled
the reduction in cell number, indicating that protein synthesis per cell was unchanged.
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We then assessed the interaction of the effects of TFA with those of an
osteoblast mitogen, in this case rat amylin (Fig. 2A). The
hydrochloride salt produced a clear stimulation of osteoblast proliferation. However, the TFA salt produced only an upward trend in
cell number, which did not reach statistical significance (Fig. 2A). Similar results were produced
with calcitonin gene-related peptide
(108 M), which increased
cell number by 8% when used as a TFA salt but by 18% when used as the
hydrochloride (Fig. 2B). Similar
experiments were performed with rat calcitonin. When formulated as the
hydrochloride salt it had no effect on osteoblast proliferation, but in
the TFA form it significantly reduced cell number (Fig.
2C). Studies of the addition of HCl
alone showed no effect on cell number (e.g., control, 2.66 ± 0.04 × 104 cells/well; HCl, 3 × 10
7 M, 2.5 ± 0.13 × 104 cells/well).
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Figure 3 shows similar experiments with the weaker
osteoblast mitogen amylin-(18). This peptide as a hydrochloride salt
significantly stimulated both cell number and thymidine incorporation,
although as the TFA salt it had no effect on either parameter.
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To determine whether the effect of TFA was specific to osteoblasts, we
repeated the studies in primary cultures of chondrocytes (Fig.
4). As with osteoblasts, there was a
concentration-dependent inhibition of thymidine incorporation and a
reduction in cell number.
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Finally, we assessed the effects of TFA in an organ culture model, the
neonatal mouse calvaria. TFA reduced thymidine incorporation in this
model, as did the TFA salt of amylin-(18) (Fig.
5). However, as in the isolated
osteoblasts, the hydrochloride salt of amylin-(1
8) stimulated
thymidine incorporation. In mouse calvaria, we also studied
45Ca release from prelabeled
bones, an index of osteoclastic bone resorption. Release of
45Ca was unaffected by TFA in
concentrations of up to 2 × 10
7 M, and amylin inhibited
bone resorption whether it was added as the TFA salt or the
hydrochloride salt (Fig. 6).
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DISCUSSION |
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The present studies establish that TFA in concentrations of the order
of 108 to
10
7 M reduces cell
proliferation in cultured osteoblasts, chondrocytes, and neonatal mice
calvariae. Thus the effect is not specific to one cell type or to one
species of origin, and it is seen in both cell and tissue culture. This
raises the possibility that it may be a general phenomenon that should
be considered when the effects of any purified peptide are being
assessed in any culture system. It is probably only relevant when the
peptide is being added at concentrations of
>10
9 M, because lower
peptide levels will result in TFA concentrations that are below those
found to have effects in the present studies. However, it is possible
that cell types may vary in their sensitivity to TFA, so it is an issue
that should be addressed in any culture system used to assess the
effects of purified or synthetic proteins.
The consequences of failing to take into account the effects of the
accompanying anion of a peptide salt are demonstrated in the present
study. Weak proliferative effects can be overlooked because they are
counteracted by the TFA contaminating the peptide [as with
amylin-(18) in the present studies], or a peptide that has no
effect on proliferation might be found to apparently inhibit cell
growth, as we found with calcitonin. These effects, however, seem to be
specific to cell replication because protein synthesis was only reduced
in parallel with cell number and cell-mediated bone resorption was not
affected by TFA. These two negative findings do not provide any
guarantee that all other cellular processes are not influenced by TFA,
and the onus remains on investigators to establish this fact for the
end points being assessed in a given study. The use of vehicle controls
has not addressed this issue to date, because virtually no one has been
adding TFA to their control cultures because of a general unawareness
of the presence of this contaminant in purified peptides.
The mechanisms of the difference in effects of TFA and hydrochloride
salts remain to be determined. They clearly are effects of TFA and not
of HCl, because they are seen when TFA is added alone and because the
addition of 107 M
H+ or
Cl
ions could not produce
such biological effects. This amount of H+ ion is well within the
buffering capacity of the media.
Cl
is the principal anion
of the media and is already present in a concentration of more than 100 mM.
The biological effects of TFA have already been investigated in two other contexts. TFA is the principal metabolite of the general anesthetic agent halothane. In vivo, it causes the formation of trifluoroacetylated forms of many proteins, a process thought to underlie the development of halothane hepatitis (4). Effects have also been found in cultures of glioma cells and are suggested to underlie halothane-associated neurotoxicity (7). This widespread modification of cell proteins could underlie the effects found in the present study. Possibly related to this effect is the fact that TFA tends to break down the intermolecular structure of water; that is, it is a chaotropic anion. Such anions have been shown to impact on membrane function, enzymatic catalysis, the secondary structure of proteins, and protein stability (1). These actions provide potential explanations for the suppression of cell proliferation observed in the present studies.
In conclusion, we have demonstrated significant biological effects of
TFA, which is present as a contaminant in many commercially available
peptides. This finding casts doubt on the results of any studies of
purified peptides in concentrations above
109 M in which measures
were not taken to remove TFA from the peptides used or to add it in
appropriate concentrations to controls. Fortunately, the problem is
easily circumvented by the production of hydrochloride salts from the
TFA salts generally available.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. P. Wiggins for helpful discussions regarding this work and to Dr. C. A. Poole for advice regarding chondrocyte preparations.
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FOOTNOTES |
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This work was funded by the Health Research Council of New Zealand and the Endocore Trust.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. Cornish, Dept. of Medicine, Univ. of Auckland, Private Bag 92019, Auckland 1001, New Zealand (E-mail: j.cornish{at}auckland.ac.nz).
Received 18 September 1998; accepted in final form 14 June 1999.
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