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J Dent Res 83(1): 22-26, 2004
© 2004 International and American Associations for Dental Research

RESEARCH REPORT
Biomaterials & Bioengineering

Salivary Esterase Activity and Its Association with the Biodegradation of Dental Composites

Y. Finer1, and J.P. Santerre2,*

1 Restorative Discipline and
2 Biomaterials Discipline, Faculty of Dentistry, University of Toronto, Toronto, ON, Canada M5G 1G6;

* corresponding author, paul.santerre{at}utoronto.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pseudocholinesterase (PCE) and cholesterol esterase (CE) can hydrolyze bisphenylglycidyl dimethacrylate (bisGMA) and triethylene glycol dimethacrylate (TEGDMA) monomers. This study will test the hypothesis that enzyme activities showing CE and PCE character are found in human saliva at levels sufficient to hydrolyze ester-containing composites important to restorative denstistry. The study also seeks to ask if the active sites of CE and PCE with respect to composite could be inhibited. Photo-polymerized model composite resin was incubated in PCE and CE solutions, in the presence and absence of a specific esterase inhibitor, phenylmethylsulfonyl fluoride (PMSF). Incubation solutions were analyzed for resin degradation products by high-performance liquid chromatography (HPLC), UV spectroscopy, and mass spectrometry. Saliva was found to contain both hydrolase activities at levels that could degrade composite resins. PMSF inhibited the composite degradation, indicating a material hydrolysis mechanism similar to the enzymes’ common function.

KEY WORDS: biodegradation • biomaterial hydrolysis • esterases • serine esterase inhibitor • dental resins


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human saliva is a complex mixture of liquid and particulate matter which originates from several sources (Chauncey, 1961; Nakamura and Slots, 1983). One class of salivary components that has some interest in terms of composite biodegradation is the esterases (Lindqvist et al., 1977).

Cholinesterases (ChE) consist of a group of esterases that hydrolyze choline esters at a higher rate than they do other esters (Ryhanen et al., 1983). Various types of ChE can be differentiated by the use of either specific substrates or selective inhibitors. In humans, two main types of ChE exist: acetylcholinesterase and pseudocholinesterase (PCE) (Ryhanen et al., 1983). More recently, ChE activity with respect to TEGDMA has been reported (Yourtee et al., 2001).

Mononuclear phagocytic cells, i.e., macrophages and monocytes, produce esterases and are present in normal and inflamed gingiva (Payne et al., 1975; Tenovuo, 1990; Lappin et al., 1999). Most of the esterase-related activity in mature macrophages is cholesterol esterase (CE) (Li and Hui, 1997). Cholesterol esterase generation increases in macrophages under a variety of conditions (Lindhorst et al., 1997; Labow et al. 2001).

It has been shown that both CE and PCE can hydrolyze the synthetic matrix components of commercial and model composite resin systems (Santerre et al., 1999, 2001). To determine the concentrations of such activities in human saliva, we profiled esterase activities using o- and p-isomers of nitrophenylacetate and nitrophenylbutyrate (Labow et al., 1994), as well as a PCE-specific substrate, butyrylthiocholine iodide (BTC).


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Model Composite Resin Samples
Model composite resins were synthesized as described before (Shajii and Santerre, 1999). Briefly, bisGMA and TEGDMA (kindly supplied by Esschem, Linwood, PA, USA) were used as the matrix phase with a weight ratio of 55/45 [based on material safety data sheets (MSDS) of commercial restorative composite resins]. A silanated barium glass filler (kindly supplied by L.D. Caulk/DENTSPLY, Milford, DE, USA), 1-µm average diameter, was added to the resin mixture at 60% weight fraction of the composite resin’s total mass. Camphorquinone (0.3 wt%) and 2-(dimethyl amino) ethyl methacrylate (0.1 wt%) were used as the photo-activating system. The composite resin was de-gassed in a vacuum oven (-760 mm Hg gauge pressure, 30°C) overnight and stored at 4°C until required.

Prior to the preparation of the cured composite resin samples, the composite pastes were warmed at room temperature for 1 hr. For sample preparation, this material was photo-polymerized into cylindrical pellets (4 mm height x 4 mm diameter) as previously described (Jaffer et al., 2002).

Enzyme Preparation
We prepared CE (Item No. 70-1081-01, Lot No. 9750, Genzyme, Cambridge, MA, USA) and PCE (C-5386, Sigma, St. Louis, MO, USA) by dissolving the enzymes at the desired concentrations (see below for specific experiments) in phosphate-buffered saline (D-PBS, 21600-010, Gibco, Grand Island, NY, USA). All solutions were sterile-filtered with the use of a 0.22-µm filter (Millex®-GP, 0.22 µm Filter unit, Cat. No. SLGPR25LS, Millipore, Bedford, MA, USA). The prepared CE and PCE solutions used for replenishing enzyme activity in the biodegradation experiments were stored at -80°C. One unit of CE activity was defined as a change of absorbance of 0.01 optical density (OD) per min at 410 nm with para-nitrophenylacetate (p-NPA) as a substrate at pH 7.0 and 25°C (Labow et al., 1994). We selected this definition of activity to allow comparisons to be made with previous degradation studies that used a similar definition of units (Santerre et al., 1999). We determined the PCE activity for this study by measuring changes in OD at a wavelength of 405 nm, using butyrylthiocholine iodide (BTC) as a substrate [cholinesterase (BTC) activity kit, Sigma, Procedure No. 421]. For PCE, a unit of enzyme activity was defined as 1 mmol butyrate released per 1 mL enzyme solution per min. The spectrophotometer unit was an Ultrospec® II (LKB Biochrom, Cambridge, England).

Enzyme Substrate Specificities
We prepared the nitrophenyl-isomers—o-nitrophenylacetate (o-NPA) (N-9001 Sigma, St. Louis, MO, USA), p-nitrophenylacetate (p-NPA) (N-8130, Sigma), o-nitrophenylbutyrate (o-NPB) (N-9751, Sigma), and p-nitrophenylbutyrate (p-NPB) (N-9876, Sigma)—by dissolving each agent in 1 mL methanol, which was then diluted with 100 mL of 0.1 M sodium acetate, pH 5.0, to yield a final concentration of 1 mM. We determined CE and PCE activities by incubating the enzymes in a solution containing 1.0 mL of 0.05 M phosphate buffer, pH 7.0, and 0.5 mL of the prepared nitrophenyl ester substrate solution. We took spectrophotometric measurements at 410 nm, as described above, to measure the unit of activity per µg protein. The enzymes were also assayed with BTC as a substrate as described above.

Hydrolase Activity in Human Saliva
Unstimulated whole human saliva (human ethics protocol approved by the Univ. of Toronto) was collected into 50-mL centrifuge tubes from seven healthy individuals and immediately stored on ice before being processed according to a method slightly modified from that previously reported (Munksgaard and Freund, 1990). Bulk debris was separated from whole saliva by centrifugation (Centrifuge international equipment Co., Needham, MA, USA) at 2400 RPM for 10 min at 4°C. The supernatant was collected and then filtered via 0.22-µm syringe filters (Millex®-GP, 0.22 µm Filter unit, Cat. No. SLGPR25LS, Millipore, Bedford, MA, USA). Aliquots of the filtered saliva were tested for hydrolase activity and compared with the stock CE and PCE enzymes with the use of 5 substrates—p-NPA, o-NPA, p-NPB, o-NPB, and BTC—as described above. The experiment was run with triplicate sample groups.

Inhibition of CE and PCE with PMSF
Prior to the biodegradation experiments, we measured the activity of the enzymes with and without the esterase inhibitor, phenylmethyl sulfonyl fluoride (PMSF) (P-7626, Sigma, St. Louis, MO, USA), by adding the inhibitor, dissolved in ethanol, to the enzyme solutions prior to their activity measurement (Labow et al., 1994). We also assayed the enzymes with the same volume of ethanol (PMSF carrier solvent) without the inhibitor to assess if this carrier solvent influenced the enzyme’s activity. PMSF dissolved in ethanol was also used as a non-enzyme control. The final concentration range of PMSF was adjusted to 1 mM in the CE solution (1 unit/mL) and 0.5 mM for PCE (1 unit/mL). These concentrations were established to provide approximately 60% inhibition values relative to their respective substrates.

Before the biodegradation experiment, the cured composite samples were pre-incubated in D-PBS for 48 hrs at 37°C to remove a significant fraction of the unreacted leachable monomers (Tanaka et al., 1991). Following pre-incubation, three cured pellets for each condition were placed in 2-mL sterile vials. The total surface area of the samples for each of these groups was 2.26 cm2. Each group was incubated (37°C and pH 7.0) in 1 mL of either buffer, CE, or PCE solution (n = 3). Ethanol alone (as a control) or PMSF dissolved in ethanol was added to either buffer, CE, or PCE replenishing solutions prior to their addition to the incubation solutions. A CE and PCE concentration of 0.1 unit/mL was chosen for the incubation, since the saliva analysis studies indicated that the esterase activity based on p-NPA substrate was about 0.1 unit/mL in saliva (see RESULTS). Accordingly, PMSF concentrations were scaled to 0.1 mM and 0.05 mM, respectively, for CE and PCE. The composite resin biodegradation experiment was run for 16 days with daily enzyme replenishment. The collected incubation solutions were filtered by means of a Millipore centrifuge filter device (Ultrafree®-CL, UFC4LCC00 5000 NMWL, Millipore, Bedford, MA, USA) and a centrifuge (Centrifuge international equipment Co., Needham, MA, USA) at 2400 RPM and kept refrigerated at 4°C until required for chromatographic analysis.

Product Isolation by High-performance Liquid Chromatography (HPLC)
A WatersTM HPLC system (Waters, Mississauga, ON, Canada) was used for the chromatographic separation of the degradation products. Specifically, the analyses of methacrylic acid (MA) derived from TEGDMA and bisGMA and bishydroxy propoxy phenyl propane (bisHPPP) derived from bisGMA were of interest. A Phenomenex Luna 5 µm C18 4.6 x 250 (Phenomenex, Torrance, CA, USA) column was used to separate and isolate the products. The mobile phase consisted of HPLC-grade methanol (Code 6701-7, Lot 34955, Caledon Laboratories LTD, Georgetown, ON, Canada) and a 2-mM buffer solution of ammonium acetate (37 233-1, Aldrich, Milwaukee, WI, USA). The pH of the buffer was adjusted to 3.0 with hydrochloric acid 6.00 N (VW3204-1, VWR, West Chester, PA, USA).

The HPLC fractions of interest were collected and then analyzed by mass spectrometry via a Perkin-Elmer/Sciex (Concord, ON, Canada) API-III triple-quadrupole mass spectrometer (LC/MS/MS) located in the Carbohydrate Research Center, University of Toronto, Ontario, Canada.

Statistical Analysis
For the enzyme substrate specificities, hydrolase activity in human saliva, and inhibition of the enzymes’ activity experiments, a Scheffé multiple comparison after one-way analysis of variance was applied for each experiment. The results were expressed as a mean ± standard error. For the enzyme substrate specificities, a factorial analysis was performed for the length and position of the side-chain.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Enzyme Substrate Specificities
Fig. 1Go depicts the results for enzyme activity with the nitrophenyl esters and BTC substrates. The CE preparation contained from 10 to 314 times more activity than the PCE solutions, depending on the specific nitrophenyl substrate used (Fig. 1Go). However, what was of much more interest was the specific pattern of activity shown toward the different substrates. The difference in CE activity when the shorter chain acetate substrates and their analogous longer chain butyrate were compared was significant and different from that observed for PCE (p < 0.05) (Fig. 1Go). The difference in PCE activity associated with the para-isomers of the different esters vs. the ortho-isomers of the esters was greater than the difference observed for the same substrates with CE (p < 0.05) (Fig. 1Go). In contrast to PCE, CE showed no activity with the BTC substrate, despite the significant esterase activity shown with the nitrophenyl substrates (Fig. 1Go).



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  		Figure 1. Activity profiles for CE (A) and PCE (B) with para-nitrophenylacetate (p-NPA), ortho-nitrophenylacetate (o-NPA), para-nitrophenylbutyrate (p-NPB), ortho-nitrophenylbutyrate (o-NPB), and butyrylthiocholine iodide (BTC). All data are reported as mean ± standard error. N = 3. Standard deviations for CE range from 0.01 to 0.16 units/µg protein, and for PCE from 0.0002 to 0.003 units/µg protein.

 
Hydrolase Activity in Human Saliva
Hydrolase activities from human saliva were measured with the use of nitrophenyl ester substrates and compared with the stock CE (Fig. 2Go). All subjects exhibited esterase activity when analyzed with the different nitrophenyl substrates. The average activity level measured with p-NPA as a substrate was 0.19 ± 0.02 unit/mL, with a range between 0.09 and 0.26 unit/mL (Fig. 2Go). The highest measured activity for all subjects was observed with p-NPB, with an average of 0.59 ± 0.18 unit/mL and a range between 0.14 unit/mL and 1.48 units/mL (Fig. 2Go). All subjects demonstrated esterase PCE-like activity when analyzed with butyrylthiocholine (BTC). The average activity level measured with BTC as a substrate was 0.011 ± 0.001 unit/mL, with a range between 0.004 and 0.018 unit/mL.



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  		Figure 2. Esterase-like activity measured in human saliva, collected from different subjects and measured with para-nitrophenylacetate (p-NPA), ortho-nitrophenylacetate (o-NPA), para-nitrophenylbutyrate (p-NPB), and ortho-nitrophenylbutyrate (o-NPB), (pH 7.0 at 25°C).

 
Inhibition of CE and PCE with PMSF
The addition of ethanol (the inhibitor’s carrier solvent) to the assay solution had no significant effect on the activity of the enzyme (Fig. 3Go). The addition of PMSF to the CE and PCE solutions showed a decrease in the relative enzymatic activity of the enzyme, to 63 ± 0.5% (p < 0.05) and 58 ± 4.7% (p < 0.05), respectively (Fig. 3Go). PMSF dissolved in ethanol was used as a negative control and exhibited no activity with respect to p-NPA.



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  		Figure 3. The inhibition effect of PMSF (at PMSF concentrations to achieve 40% reduction in activity with respect to the substrates below) on the activities of CE and PCE (pH 7.0 at 25°C). The activities were measured with para-nitrophenylacetate and butyrylthiocholine as substrates for CE and PCE, respectively. PMSF concentration for CE was 1 mM and for PCE was 0.5 mM. All data are reported as mean ± standard error. N = 3. Standard deviations for CE-incubated groups range from 5.2 to 7.9%, and for PCE groups from 3.8 to 5.3%.

 
In the biodegradation experiments, the addition of PMSF to the buffer solutions had no effect on the biodegradation of the samples when compared with the buffer alone (Fig. 4Go). However, the addition of PMSF to the CE- and PCE-incubated samples produced a significant reduction in the relative amount of MA (Fig. 4AGo) (p < 0.05) generated from the residual methacrylate groups within the polymer matrix and bisHPPP product (Fig. 4BGo) (p < 0.05).



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  		Figure 4. Inhibition of CE and PCE catalyzed biodegradation for cured composite resin samples by PMSF, following 16 days’ incubation (pH 7.0 at 37°C). (A) Inhibition of methacrylic acid production. (B) Inhibition of bisHPPP production. PMSF concentration for CE was 0.1 mM and for PCE was 0.05 mM. All data are reported as mean ± standard error. N = 3. Standard deviations for MA-analyzed product range from 5.9 to 8.3%, and for bisHPPP products from 4.0 to 11.8%.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
PMSF is a serine esterase inhibitor, which alkylates the hydroxyl of the active serine site in the esterases (Sutton et al., 1986, 1990; Labow et al., 1994). Its function on both enzymes is shown in Fig. 3Go. The percent inhibition, by PMSF, on the generation of MA and bisHPPP from the composite’s polymeric matrix (Fig. 4Go) was comparable with the inhibition of the hydrolysis with the enzymes’ standard substrates (p-NPA and BTC for the respective enzymes CE and PCE) (Fig. 3Go), providing evidence to support that hydrolysis of the resin matrix possibly occurred via the same active site implicated with the more usual substrates of the enzymes (Labow et al., 1994). By establishing an initial inhibition condition that was similar for both the CE and PCE solutions (Fig. 3Go), we expected that the inhibition of the composite resin degradation would also be similar. However, this was not the case, since CE was inhibited 15–20% more than PCE with respect to the resin degradation. This would imply that the process of resin degradation exhibits greater sensitivity to CE.

The difference between the action of CE and that of PCE on the composite resin may be related to their different reactivities to natural and synthetic substrates. CE preferentially catalyzes the hydrolysis of long-chain fatty acid esters of cholesterol (Labow et al., 1983; Williams, 1985; Sutton et al., 1991; Feaster et al., 1996), while PCE catalyzes the turnover of low-molecular-weight choline esters, such as butyrylcholine. CE and PCE showed different activities with respect to the synthetic substrates, o- and p-nitrophenyl esters (Fig. 1Go). In the factorial analysis for the length and position of the ester side-chain, only the length of the side-chain was a significant variable for CE activity (p < 0.001), with a higher hydrolysis rate for the longer side-chain nitrophenyl esters. This agrees with previous reports that CE-like activity hydrolyzes the non-water-soluble chains of synthetic polyurethane (Labow et al., 1994).

The analysis of saliva with the nitrophenyl substrates suggests the presence of a strong CE-like hydrolase activity associated with the oral environment (Fig. 2Go). All human subjects showed activities toward all substrates, but the pattern of sensitivity was more similar to that of CE vs. PCE (compare Figs. 1 Goand 2Go). This was demonstrated by the slightly higher specificity toward the para-isomer vs. other isomers and the selectivity of p-NPB over that of all other substrates for all subjects and the stock CE enzyme (Labow et al., 1994). The average activity level, measured with p-NPA as a substrate, was 0.19 ± 0.02 unit/mL. In previous work, lower CE activity levels, as measured with p-NPA, have been shown to degrade composite resin samples significantly (Shajii and Santerre, 1999; Finer and Santerre, 2003). Hence, CE-like activity is present in human saliva in levels high enough to warrant concern over the biodegradation of composite monomers.

The presence of PCE-like activity in human saliva was also confirmed, with an average activity level of 0.011 ± 0.001 unit/mL. Similar results, with respect to the PCE activity levels in saliva, were found by others (Ryhanen et al., 1983; Ryhanen, 1983; Yamalik et al., 1990, 1991).

In summary, these results support that CE and PCE are suitable models for salivary esterase activity which can catalyze the hydrolysis of composite resins in the oral cavity (Jaffer et al., 2002).


   ACKNOWLEDGMENTS
 
This study was supported by the Natural Science and Engineering Research Council Of Canada (NSERC) and the Alpha Omega Foundation of Canada.

Received November 11, 2002; Last revision September 18, 2003; Accepted September 19, 2003


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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
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