RESEARCH REPORTS
Biomaterials & Bioengineering
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@utoronto.ca
J Dent Res 83(1):22-26, 2004
ABSTRACT
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.
Received November 11, 2002; Last revision September 18,
2003; Accepted September 19, 2003
22
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International and American Associations for Dental Research
Salivary Esterase Activity and Its
Association with the
Biodegradation of Dental
Composites
INTRODUCTION
H uman 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
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-␮m 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
J Dent Res 83(1) 2004 Human Saliva Esterase Activity and Composite Resins 23

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-␮m filter (Millex®-GP, 0.22
␮ m 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 Figure 1. Activity profiles for CE (A) and PCE (B) with para-nitrophenylacetate ( p -NPA), ortho-
stored at -80°C. One unit of CE nitrophenylacetate (o-NPA), para-nitrophenylbutyrate (p-NPB), ortho-nitrophenylbutyrate (o-NPB), and
activity was defined as a change of 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/␮g protein, and for PCE from 0.0002 to 0.003
absorbance of 0.01 optical density units/␮g protein.
(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 Inhibition of CE and PCE with PMSF
this definition of activity to allow comparisons to be made with
Prior to the biodegradation experiments, we measured the activity
previous degradation studies that used a similar definition of units
of the enzymes with and without the esterase inhibitor,
(Santerre et al., 1999). We determined the PCE activity for this
phenylmethyl sulfonyl fluoride (PMSF) (P-7626, Sigma, St. Louis,
study by measuring changes in OD at a wavelength of 405 nm,
MO, USA), by adding the inhibitor, dissolved in ethanol, to the
using butyrylthiocholine iodide (BTC) as a substrate
enzyme solutions prior to their activity measurement (Labow et al.,
[cholinesterase (BTC) activity kit, Sigma, Procedure No. 421]. For
1994). We also assayed the enzymes with the same volume of
PCE, a unit of enzyme activity was defined as 1 mmol butyrate
ethanol (PMSF carrier solvent) without the inhibitor to assess if
released per 1 mL enzyme solution per min. The
®
this carrier solvent influenced the enzyme's activity. PMSF
spectrophotometer unit was an Ultrospec II (LKB Biochrom,
dissolved in ethanol was also used as a non-enzyme control. The
Cambridge, England).
final concentration range of PMSF was adjusted to 1 mM in the CE
Enzyme Substrate Specificities solution (1 unit/mL) and 0.5 mM for PCE (1 unit/mL). These
We prepared the nitrophenyl-isomers—o-nitrophenylacetate (o- concentrations were established to provide approximately 60%
NPA) (N-9001 Sigma, St. Louis, MO, USA), p-nitrophenylacetate inhibition values relative to their respective substrates.
(p-NPA) (N-8130, Sigma), o-nitrophenylbutyrate (o-NPB) (N- Before the biodegradation experiment, the cured composite
9751, Sigma), and p-nitrophenylbutyrate (p-NPB) (N-9876, samples were pre-incubated in D-PBS for 48 hrs at 37°C to remove
Sigma)—by dissolving each agent in 1 mL methanol, which was a significant fraction of the unreacted leachable monomers (Tanaka
then diluted with 100 mL of 0.1 M sodium acetate, pH 5.0, to yield et al., 1991). Following pre-incubation, three cured pellets for each
a final concentration of 1 mM. We determined CE and PCE condition were placed in 2-mL sterile vials. The total surface area
activities by incubating the enzymes in a solution containing 1.0 of the samples for each of these groups was 2.26 cm2. Each group
mL of 0.05 M phosphate buffer, pH 7.0, and 0.5 mL of the was incubated (37°C and pH 7.0) in 1 mL of either buffer, CE, or
prepared nitrophenyl ester substrate solution. We took PCE solution (n = 3). Ethanol alone (as a control) or PMSF
spectrophotometric measurements at 410 nm, as described above, dissolved in ethanol was added to either buffer, CE, or PCE
to measure the unit of activity per ␮g protein. The enzymes were replenishing solutions prior to their addition to the incubation
also assayed with BTC as a substrate as described above. solutions. A CE and PCE concentration of 0.1 unit/mL was chosen
for the incubation, since the saliva analysis studies indicated that the
Hydrolase Activity in Human Saliva esterase activity based on p-NPA substrate was about 0.1 unit/mL
Unstimulated whole human saliva (human ethics protocol approved in saliva (see RESULTS). Accordingly, PMSF concentrations were
by the Univ. of Toronto) was collected into 50-mL centrifuge tubes scaled to 0.1 mM and 0.05 mM, respectively, for CE and PCE. The
from seven healthy individuals and immediately stored on ice before composite resin biodegradation experiment was run for 16 days
being processed according to a method slightly modified from that with daily enzyme replenishment. The collected incubation
previously reported (Munksgaard and Freund, 1990). Bulk debris solutions were filtered by means of a Millipore centrifuge filter
was separated from whole saliva by centrifugation (Centrifuge device (Ultrafree®-CL, UFC4LCC00 5000 NMWL, Millipore,
international equipment Co., Needham, MA, USA) at 2400 RPM for Bedford, MA, USA) and a centrifuge (Centrifuge international
10 min at 4°C. The supernatant was collected and then filtered via equipment Co., Needham, MA, USA) at 2400 RPM and kept
0.22-␮m syringe filters (Millex®-GP, 0.22 ␮m Filter unit, Cat. No. refrigerated at 4°C until required for chromatographic analysis.
SLGPR25LS, Millipore, Bedford, MA, USA). Aliquots of the
filtered saliva were tested for hydrolase activity and compared with Product Isolation by High-performance Liquid
the stock CE and PCE enzymes with the use of 5 substrates—p- Chromatography (HPLC)
NPA, o-NPA, p-NPB, o-NPB, and BTC—as described above. The A WatersTM HPLC system (Waters, Mississauga, ON, Canada) was
experiment was run with triplicate sample groups. used for the chromatographic separation of the degradation

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International and American Associations for Dental Research

24
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).
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 ␮m 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
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%.
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International and American Associations for Dental Research
Finer & Santerre J Dent Res 83(1) 2004
(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
Enzyme Substrate Specificities
Fig. 1 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. 1). 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. 1). 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. 1). In contrast to PCE, CE showed no
activity with the BTC substrate, despite the significant esterase
activity shown with the nitrophenyl substrates (Fig. 1).
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. 2). 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.
2). 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. 2). 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.
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. 3). 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),
J Dent Res 83(1) 2004 Human Saliva Esterase Activity and Composite Resins 25

respectively (Fig. 3). PMSF

dissolved in ethanol was used as a
negative control and exhibited no
activity with respect to p-NPA.
In the biodegradation experi-
ments, the addition of PMSF to
the buffer solutions had no effect
on the biodegradation of the
samples when compared with
the buffer alone (Fig. 4).
However, the addition of PMSF
to the CE- and PCE-incubated
samples produced a significant
reduction in the relative amount
of MA (Fig. 4A) (p < 0.05) 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
generated from the residual of bisHPPP production. PMSF concentration for CE was 0.1 mM and for PCE was 0.05 mM. All data are
methacrylate groups within the reported as mean + standard error. N = 3. Standard deviations for MA-analyzed product range from 5.9
polymer matrix and bisHPPP to 8.3%, and for bisHPPP products from 4.0 to 11.8%.
product (Fig. 4B) (p < 0.05).

DISCUSSION
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. 3. The percent inhibition, by PMSF, on the
generation of MA and bisHPPP from the composite's polymeric
matrix (Fig. 4) 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. 3),
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. 3), 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. 1). 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. 2). 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 and 2). 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.
REFERENCES
Chauncey HH (1961). Salivary enzymes. J Am Dent Assoc 63:360-
368.
Feaster SR, Lee K, Baker N, Hui DY, Quinn DM (1996). Molecular
recognition by cholesterol esterase of active site ligands: structure-
reactivity effects for inhibition by aryl carbamates and subsequent
carbamylenzyme turnover. Biochemistry 35:16723-16734.
Finer Y, Santerre JP (2003). Biodegradation of a dental composite by
esterase- dependence on enzyme concentration and specificity. J
Biomater Sci Polymer Ed 14:837-849.
Jaffer F, Finer Y, Santerre JP (2002). Interactions between resin
monomers and commercial composite resins with human saliva
derived esterases. Biomaterials 23:1707-1719.
Labow RS, Adams KA, Lynn KR (1983). Porcine cholesterol esterase,

Downloaded from jdr.sagepub.com at Umea University Library on April 5, 2015 For personal use only. No other uses without permission.

International and American Associations for Dental Research

26
a multiform enzyme. Biochim Biophys Acta 749:32-41.
Labow RS, Duguay DG, Santerre JP (1994). The enzymatic hydrolysis
of a synthetic biomembrane: a new substrate for cholesterol and
carboxyl esterases. J Biomater Sci Polymer Ed 6:169-179.
Labow RS, Meek E, Santerre JP (2001). Model systems to assess the
destructive potential of human neutrophils and monocyte-derived
macrophages during the acute and chronic phases of inflammation.
J Biomed Mater Res 54:189-197.
Lappin DF, Koulouri O, Radvar M, Hodge P, Kinane DF (1999).
Relative proportions of mononuclear cell types in periodontal
lesions analyzed by immunohistochemistry. J Clin Periodontol
26:183-189.
Li F, Hui DY (1997). Modified low density lipoprotein enhances the
secretion of bile salt-stimulated cholesterol esterase by human
monocyte-macrophages. species-specific difference in macrophage
cholesteryl ester hydrolase. J Biol Chem 272:28666-28671.
Lindhorst E, Young D, Bagshaw W, Hyland M, Kisilevsky R (1997).
Acute inflammation, acute phase serum amyloid A and cholesterol
metabolism in the mouse. Biochim Biophys Acta 1339:143-154.
Lindqvist L, Nord CE, Söder PO (1977). Origin of esterases in human
whole saliva. Enzyme 22:166-175.
Munksgaard EC, Freund M (1990). Enzymatic hydrolysis of
(di)methacrylates and their polymers. Scand J Dent Res 98:261-
267.
Nakamura M, Slots J (1983). Salivary enzymes. Origin and
relationship to periodontal disease. J Periodontal Res 18:559-569.
Payne WA, Page RC, Ogilvie AL, Hall WB (1975). Histopathologic
features of the initial and early stages of experimental gingivitis in
man. J Periodontal Res 10:51-64.
Ryhanen RJ (1983). Pseudocholinesterase activity in some human
body fluids. Gen Pharmacol 14:459-460.
Ryhanen R, Närhi M, Puhakainen E, Hanninen O, Kontturi-Närhi V
(1983). Pseudocholinesterase activity and its origin in human oral
fluid. J Dent Res 62:20-23.
Santerre JP, Shajii L, Tsang H (1999). Biodegradation of commercial
dental composites by cholesterol esterase. J Dent Res 78:1459-
1468.
Downloaded from jdr.sagepub.com at Umea University Library on April 5, 2015 For personal use only. No other uses without permission.
International and American Associations for Dental Research
Finer & Santerre J Dent Res 83(1) 2004
Santerre JP, Shajii L, Leung BW (2001). Relation of dental composite
formulations to their degradation and the release of hydrolyzed
polymeric-resin-derived products. Crit Rev Oral Biol Med 12:136-
151.
Shajii L, Santerre JP (1999). Effect of filler content on the profile of
released biodegradation products in micro-filled bis-
GMA/TEGDMA dental composite resins. Biomaterials 20:1897-
1908.
Sutton LD, Stout JS, Hosie L, Spencer PS, Quinn DM (1986). Phenyl-
n-butylborinic acid is a potent transition state analog inhibitor of
lipolytic enzymes. Biochem Biophys Res Commun 134:386-392.
Sutton LD, Lantz JL, Eibes T, Quinn DM (1990). Dimensional
mapping of the active site of cholesterol esterase with alkylboronic
acid inhibitors. Biochim Biophys Acta 1041:79-82.
Sutton LD, Froelich S, Hendrickson HS, Quinn DM (1991).
Cholesterol esterase catalyzed hydrolysis of mixed micellar
thiophosphatidylcholines: a possible charge-relay mechanism.
Biochemistry 30:5888-5893.
Tanaka K, Taira M, Shintani H, Wakasa K, Yamaki M (1991).
Residual monomers (TEGDMA and Bis-GMA) of a set visible-
light-cured dental composite resin when immersed in water. J
Oral Rehabil 18:353-362.
Tenovuo J (1990). Patients with locally and generally reduced host
defence. J Clin Periodontol 17:525-526.
Williams FM (1985). Clinical significance of esterases in man. Clin
Pharmacokinet 10:392-403.
Yamalik N, Ozer N, Caglayan F, Caglayan G (1990). Determination of
pseudocholinesterase activity in the gingival crevicular fluid,
saliva, and serum from patients with juvenile periodontitis and
rapidly progressive periodontitis. J Dent Res 69:87-89.
Yamalik N, Ozer N, Caglayan F, Caglayan G, Akdoganli T (1991).
The effect of periodontal therapy on salivary pseudocholinesterase
activity. J Dent Res 70:988-990.
Yourtee DM, Smith RE, Russo KA, Burmaster S, Cannon JM, Eick
JD, et al. (2001). The stability of methacrylate biomaterials when
enzyme challenged: kinetic and systematic evaluations. J Biomed
Mater Res 57:522-531.