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JOURNAL OF I=NDODONTICS
Copyright 1993 by The American Association of Endodontists

Printed in U.S.A.

VOL. 19, NO. 2, FEBRUARY1993

Effect of Calcium Hydroxide on Bacterial

Lipopolysaccharide
Kamran E. Safavi, DMD, MED, and Frank C. Nichols

Apical periodontitis and its concomitant periapical

osteolysis is caused by pulpal infection, and bacterial lipopolysaccharide (LPS) is known to play a
major role in the bone resorption process. Little is
known concerning the effect of root canal intervisit
dressings on residual LPS in root canals after bacterial cell lysis. The purpose of this study was to
evaluate the effects of calcium hydroxide on bacterial LPS. Free hydroxy fatty acids were quantified
in samples of LPS treated with calcium hydroxide.
Calcium hydroxide treatment of LPS was shown to
release elevated quantities of hydroxy fatty acids. It
was concluded that calcium hydroxide hydrolyzed
the lipid moiety of bacterial LPS, resulting in the
release of free hydroxy fatty acids. This result suggests that calcium hydroxide-mediated degradation
of LPS may be an important reason for the beneficial
effects obtained with calcium hydroxide use in clinical endodontics.

result of bacterial cell lysis (9). Any LPS remaining in the root
canals can potentially affect periapical tissues during root
canal infection and after root canal bacteria are rendered
nonviable.
It is shown that monocytes and other immune cells have
an exquisite sensitivity to bacterial LPS, and as a result, at
minute concentrations, LPS may exert significant effects on
the host tissues (2). Residual LPS in the root canals, therefore,
may have major clinical consequences in endodontic treatment.
The fate of the LPS in root canals, after destruction of
microorganisms, is not known. The purpose of this study was
to evaluate the effects of calcium hydroxide on bacterial LPS
in vitro.
MATERIALS AND METHODS
Glass test tubes containing aqueous suspensions of bacterial
LPS (Salmonella typhimurium, hot water-phenol extracted,
30 ~g/ml; Difco Laboratories Inc., Detroit, MI), and/or calcium hydroxide powder (25 mg/ml), or pyrogen-free water
were vortexed for 20 s and incubated at 37C for 7 days,
during which they were vortexed once a day for 20 s. The
contents of each tube, hereafter referred to as "samples," were
prepared and analyzed for levels of free hydroxy fatty acids,
using a modification of a method described previously (10).
Briefly, nonadecanoic acid (c19:o, 30 ng/ml; Sigma Chemical
Co., St. Louis, MO) was added to each sample and the samples
were dried by lyophilization. The dried materials were reconstituted in water and the pH was adjusted to 1 with concentrated hydrochloric acid. The samples were then rapidly extracted with chloroform (three times), and pooled extracts
were dried under a stream of N2 gas. The dried fatty acids
were dissolved in acetonitrile (30 #1) and treated with 35%
pentafluorobenzyl bromide in acetonitrile (10 ~l) and diisopropylethylamine (10 ~1). The samples were then heated for
20 min at 40C and dried under a stream of N2 gas. The
resultant pentofluorbenzyl esters were then treated with N, Obis(trimethylsilyl)trifluoracetamide (50 ~1) and incubated
overnight. All derivatizating agents were purchased from
Pierce, Rockford, IL.
The samples were analyzed using a gas chromatograph
(Hewlett Packard 5890; Hewlett-Packard Co., Avondale, PA)
equipped with HP- 1 column (Ultra- 1, 12 x 0.2 mm; HewlettPackard) and interfaced with a mass spectrometer (Hewlett

Apical periodontitis and its concomitant periapical bone resorption is caused by pulpal infection (1). The mechanisms
by which microorganisms cause apical bone resorption are
not completely understood. However, it is clearly demonstrated that Gram-negative bacterial lipopolysaccharide (LPS;
endotoxin) plays a major role in stimulating the synthesis and
release of the principal osteoclast-activating cytokines,
namely, interleukin 1 and tumor necrosis factor-a from immune cells. Bacterial LPS also stimulates host cells to release
prostaglandin E2, an eicosanoid that is shown to infuence
osteoclast-mediated bone resorption (2-4).
In necrotic pulps, Gram-negative bacteria dominate the
root canal flora and bacterial LPS is present in root canals
(5). Furthermore, osteoclast-activating factors have recently
been identified in periapical tissues (3).
Elimination of microorganisms from the pulp space, therefore, has been a major goal in clinical endodontics, and a
wide variety of antimicrobial agents have been used for this
purpose. Although bacteriocidal effects of these agents have
been the subject of many reports in the past (6-8), their effects
on bacterial LPS have not been investigated until now. LPS
is shed from the cell wall during bacterial growth and as a
76

Calcium Hydroxide, LPS Degradation

Vol. 19, No. 2, February 1993

Packard 5988A). Individual samples were applied to the HP1 column held at 100C. The samples were injected using the
splitless mode and the column temperature was increased at
2*C/min to a maximum temperature of 2400C. The mass
spectrometer was used in the negative ion chemical ionization
mode with the ion source temperature held at 100C, the
electron energy of 240 eV, and an emission current of 300
mA. Methane was used as reagent gas and was maintained at
0.5 torr. LPS fatty acid samples were compared with 2hydroxy-C~6:o and C14:0 (Matreya Inc., Pleasant Gap, PA) and
3-hydroxy-C14:o, as well as with microbial fatty acid standards
(Suppelco Inc., Bellefonte, PA) for retention time and characteristic molecular ions. Hydroxy fatty acids were quantified
using selected ion monitoring of the base peak ions and
corrected for extraction losses using the nonadecanoic acid
internal standard. The levels of the 3-hydroxy tetradecanoic
(myristic) acid (3-OH-Cl4:o) and 3-hydroxy hexanoic (palmitic) acid (3-OH-Cl6:o) were averaged and compared using
Student's t test.

77

TABLE 1. Levels of free 3-hydroxy fatty acids recovered from

calcium hydroxide-treated LPS samples and control samples*

Controls
3-Hydroxy
Fatty Acid

LPS-Treated
Ca(OH)2
(ng/ml)

LPS
(ng/ml)

Ca(OH)2
(ng/ml)

H20
(ng/ml)

3-OH-C14:o
3-OH-C,6:o

1383 (454)
16 (7)

681 (345)
11 (9)

1 (1)
0

0
0

* The values are the mean of eight samples in each group. The standard deviations are
given in parentheses,

ng/ml
2500

2000

RESULTS
Free 3-hydroxy myristic and 3-hydroxy palmitic acids were
recovered in calcium hydroxide-treated LPS and LPS control
samples (Table 1). The 3-hydroxy myristic acid in particular
was recovered in substantial levels. Free hydroxy fatty acid
levels recovered in calcium hydroxide-treated samples were
universally higher than in LPS control samples. In the case of
free 3-hydroxy myristic acid levels, this difference was statistically significant (p < 0.001, t = 3.48, Fig. 1). Minute quantities of 3-hydroxy myristic acid were recovered in two of the
calcium hydroxide control samples (Table 1). No fatty acids
were identified in water control samples. Nonadecanoic acid
was identified in all samples (30 ng/ml).
DISCUSSION
The LPS of Gram-negative bacteria is located in the outer
membrane of the bacterial cell wall and is composed of three
distinct structural regions, the O-specific polysaccharide, the
common core, and a lipid component, called lipid A (l l).
Lipid A is responsible for many, if not all, biological activities
exhibited by bacterial LPS (2). The biological effects of LPS
such as toxicity, pyrogenicity, macrophage activation, and
complement activation are shown to be lost by the slightest
modification to the lipid A structure (2, 12).
The major constituents of lipid A are glucosamine, phosphate, and fatty acids. Among these fatty acids, 3-hydroxy
fatty acids are unique and ubiquitous to LPS (13). The 3hydroxy myristic acid constitutes a substantial portion of total
fatty acid content of Salmonella typhimurium LPS and was
used as a chemical marker in our study (2, 11, 13).
In modern endodontic practice, the emphasis on cleaning
of the root canals by instrumentation and irrigation has
reduced reliance on the use of disinfecting chemicals, which
are known to damage the host tissue cells (6). Nevertheless, it
is shown that after root canal instrumentation, if no endodontic dressing is used, any microorganisms remaining in the root
canals will proliferate between treatment visits (7, 8).
Several recent reports suggest that intracanal use of calcium
hydroxide, in lieu of disinfecting solutions, can efficiently kill

1500

1000

500

0
FIG 1. The levels of 3-hydroxy myristic acid recovered in calcium
hydroxide-treated LPS (solid bars) and LPS control samples (open
bars). The values are given in nanograms per milliliter in each of eight
experiments.

the bacteria in the root canals (14-16). Calcium hydroxide,

although not traditionally a routine intervisit root canal dressing, has been extensively used in root canals for treatment of
traumatized immature teeth with necrotic pulps, in the management of root resorption, and in nonsurgical correction of
root perforations (17). As a long-term intracanal dressing,
calcium hydroxide was shown to promote bony regeneration
in periapical lesions (17). Use of calcium hydroxide as a
routine intracanal dressing has been advocated in recent years
(14-16).
The reasons for the beneficial effects of calcium hydroxide
as an endodontic medicament are poorly understood (18). Its
effectiveness as a root canal dressing is attributed to its hydroxyl group which provides an alkaline environment. Despite low solubility, calcium hydroxide's hydroxyl ion disassociation raises the pH sufficiently to kill bacteria. Because of
its low solubility, a relatively large amount of calcium hydroxide can be packed into the root canals with little risk of
periapical irritation. The mobilization of hydroxyl ions, therefore, can continue for prolonged periods (16), and, as a result,
the duration of the effect of calcium hydroxide in the root
canals, unlike antiseptic solutions, is long (19).

78

Journal of Endodontics

Safavi and Nichols

Detoxification of LPS by degradation of lipid A has been

demonstrated using a variety of methods including deacylation with enzymes, acid hydrolysis, and treatment with alkali
(12, 20). Alkali treatment is shown to detoxify LPS by removing esterified fatty acids and altering its chemical conformation (12). According to one report, hydrolysis of LPS is
achieved with dilute sodium hydroxide and is facilitated with
ethyl alcohol or dimethyl sulfoxide in the hydrolyzing medium (l 2). These findings support our results by demonstrating that dilute alkali may release free fatty acids held in ester
linkages within the LPS macromolecule.
A relatively high recovery of 3-hydroxy myristic acid in
LPS controls is probably due to contaminating free fatty acids
that may have existed in LPS preparations. Recovery of
minute amounts of 3-hydroxy myristic acid in calcium hydroxide controls (less than 1 ng/ml; in two samples) may also
be due to contamination of calcium hydroxide preparation
with LPS.
The selection of LPS/calcium hydroxide contact time in
our experiments was based on the results of a recent clinical
study in which antimicrobial effects of calcium hydroxide
were shown to be best achieved if the canals were dressed with
calcium hydroxide for at least 7 days (15). Results of our
experiments indicate that calcium hydroxide may hydrolyze
bacterial LPS under conditions similar to those in which it is
clinically applied in the root canals. Thus, detoxification of
residual LPS in the root canals by calcium hydroxide treatment may be one of the mechanisms by which this agent
exerts its beneficial effects in clinical endodontics.
Dr. Safani is a member of the Department of Restorative Dentistry and
Endodontology and Dr. Nichols is a member of the Department of Periodontology, University of Connecticut Health Center, School of Dental Medicine,
Farmington, CT. Address requests for reprints to Dr. Kamran Safavi, Department of Restorative Dentistry and Endodontology, University of Connecticut
Health Center, 263 Farmington Avenue, Farmington, CT 06030.

References
1. Sundqvist G. Bacteriologic studies of necrotic denta~ pulps. Ume~,, Sweden: University of Ume&, University Odontological Dissertation no. 7, 1976.

2. Morrison DC, Ryan JL. Endotoxins and disease mechanisms. Ann Rev
Med 1987;38:417-32.
3. Wang C-Y, Stashenko P. Kinetics of bone-resorbing activity in developing
periapical lesions. J Dent Res 1991 ;70:1362-6.
4. Raisz LG. The role of prostaglandins in the local regulation of bone
metabolism. Prog Clin Bioi Res 1990;332:195-203.
5. Schein B, Schilder H. Endotoxin content in endodontically involved teeth.
J Endodon 1975;1:19-21.
6. Sp,~ngberg L. Endodontic medicaments. In Smith DC, Williams DF, eds.
Biocompatibility of dental materials. Boca Raton, FL: CRC Press, 1982:22356.
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mechanical root canal instrumentation in endedontic therapy. Scand J Dent
Res 1981 ;89:321-8.
8. Bystr(~m A, Sundqvist G. Bacteriologic evaluation of the effect of 0.5 per
cent sodium hypochlorite in endodontic therapy. Oral Surg 1983;55:307-12.
9. Mattsby-Baltzer I, Lindgren K, Lindholm B, Edebo L. Endotoxin shedding
by enterobacteria: free and cell-bound endotoxin differ in Limulus activity. Infect
Immun 1991 ;59:689-95.
10. Nichols FC, Peluso JF, Tempro PJ, Garrison SW, Payne JB. Prostaglandin E release from human monocytes treated with lipopolysacchaddes
isolated from Bacteroides intefmedius and Salmonella typhimurium: potentiation
by gamma interferon. Infect Immun 1991 ;59:398-406.
11. Qureshi N, Takayama K, HeIler D, Fenselau C. Position of ester groups
in the lipid A backbone of lipopolysaccharides obtained from Salmonella typhimurium. J Biol Chem 1983;258:12947-51.
12. Niwa M, Milner KC, Ribi E, Rudbach JA. Alteration of physical, chemical,
and biological properties of endotoxin by treatment with mild alkali. J Bacteriol
1969; 97:1069-77.
13. Tanamoto K. Development of new quantitative method for detection of
endotoxin by fluorescence labeling of 3-hydroxy fatty acid. Adv Exp Meal Biol
1990;256:203-13.
14. Safavi KE, Dowden WE, Introcaso JH, Langeland K. A comparison of
antimicrobial effects of calcium hydroxide and iodine-potassium iodide. J Endodon 1985;11:454-6.
15. Sjogren U, Figdor D, Sp&ngberg L, Sundqvist G. The antimicrobial
effect of calcium hydroxide as a short-term intracanal dressing. Int Ended J
1991 ;24:119-25.
16. Bystrom A, Claesson R, Sundqvist G. The antibacterial effect of camphorated paramonochlorophenol, camphorated phenol and calcium hydroxide
in the treatment of infected root canals. Ended Dent Traumato11985;1:170-5.
17. Martin DM, Crabb HSM Calcium hydroxide in root canal therapy. A
review. Br Dent J 1977;142:277-83.
18. Hasselgren G, Olsson B, Cvek M. Effects of calcium hydroxide and
sodium hypochlorite on the dissolution of necrotic porcine muscle tissue. J
Endodon 1988;14:125-7.
19. Messer HH, Chen R-S. The duration of effectiveness of root canal
medicaments. J Endodon 1984;10:240-5.
20. Munford RS, Hall CL. Detoxification of bacterial lipopotysaccharides
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