http://www.wholisticresearch.com/info/artshow.php3?artid=7


H2S is a potent Hg chelator, pulling mercury out of anything.
However, H2S itself is very toxic, and is 5 times higher in the
faeces of ulcerative colitis sufferers, causing severe inflammation
and impairs colon energy metabolism (by preventing butyrate oxidation
and impairing respiration chain cytochrome C oxidase). Detoxing H2S
happens through methylation, thus producing along the way
methylmercury, which is absorbed, and finds its way quickly to the
brain and the CNS, impairing its function in a variety of ways such
as memory problems, sleep disturbance, anxiety, depression,
moodiness, irritability, tremors, lack of concentration, tiredness,
headaches, fine tremors. Boyd Haley has shown that the changes in the
brains of patients with Alzheimer's disease, such as its effects on
nucleotide binding, are identical to the effect of mercury on brain
tissues.


Toxins Produced by Oral Microorganisms and Their Toxic Effects on
Critical Enzymes in the Human Body
By J. Curt Pendergrass Ph.D. President Affinity Labeling
Technologies, Inc.


"Poison is in everything, and no thing is without poison. The dose
makes it either a poison or a remedy"
Paracelsus

3 Types of Bacterial Toxins
Exotoxins
Endotoxins
Non-Protein Toxins

Types of Bacterial Toxins
Exotoxins
Protein toxins released extracellularly as the bacteria grows.
These toxins may travel from a focus of infection to distant parts
of the body and hence cause damage in regions far removed from the
site of microbial growth.
Examples: Diphtheria toxin (Corynebacterium diphtheriae), Cholera
toxin (Vibrio cholera), Pertussis toxin (Bordetella pertussis)

Endotoxins
Toxins which are generally bound to the cell surface and released in
large amounts only when the cells lyse.
Gram-negative bacteria produce lipopolysaccharides as part of the
outer layer of their cell walls which are toxic under many
conditions.

Non-Protein Toxins
Byproducts of bacterial metabolism


3 Classes of Non-Protein Bacterial Toxins
Volatile Sulfur Compounds (VSCs)
Hydrogen sulfide
Methyl mercaptan
Dimethyl sulfide
Dimethyl disulfide

Polyamines
Diaminobutane (Putrescine)
Diaminopentane (Cadaverine)
Spermidine

Short-Chain Carboxylic Acids (SSCAs)
Acetic acid
Propionic acid
Butyric acid


Evidence for Small Organic (Nonprotein) Bacterial Toxins
Culture medium filtrates from P. gingivalis have been shown to
inhibit collagen matrix production by chicken
embryo cartilage cells in vitro and heating of the filtrates to 100°C
did not abolish this inhibitory activity.1
Low concentrations of denatured extracts of dental plaque have been
shown to inhibit type I collagen synthesis
in cultured fetal rat calvaria suggesting that some component other
than protein was likely the causative agent.2
Metabolic byproducts in the spent medium from P. gingivalis cultures
with a molecular size under 1 kDa
were shown to inhibit bone mineralization and induced bone resorption
in fetal long-bone rudiments in vitro.3


1Touw et al., (1982). J. Periodontol. Res. 17:351-357. 2Multanen et
al., (1985). J. Periodontol.
Res. 20:637-643. 3Bom-van Noorloos et al., (1989). J. Clin.
Periodontol. 16:412-418.

Porphyromonas gingivalis, A Common Pathogen in both Periodontal and
Pulpal Infections
Porphyromonas gingivalis (representative magnification x 60,000)

Proposed Virulence Factors of Porphyromonas gingivalis
Lipopolysaccharide (endotoxin)
Proteases
Fimbriae proteins
Modulation of host immune responses
Phosphatases


Life below the gum line: pathogenic mechanisms of Porphyromonas
gingivalis.
Lamont and Jenkinson (1998). Microbiol. Mol. Biol. Rev. 62:1244-
1263.
Extracts of Oral Bacteria Are Toxic to Cells in Culture

Reasons for Study
Hypothesis: Bacteria considered periodontal pathogens or their
metabolic byproducts can inhibit osteogenesis
(bone formation) resulting in loss of alveolar bone in periodontal
disease.
Purpose: To determine the direct effects of metabolic products and
sonicated extracts of Porphyromonas
gingivalis, on bone formation in the chick periostal osteogenesis
(CPO) model.
CPO cultures enriched in osteoblast containing no osteoclast have
been demonstrated to be a reliable
model of osteogenic cell differentiation and mineralized bone
formation.

Choice of Bacteria: P. gingivalis is a microbial pathogen associated
with periodontitis.


Loomer et al., (1994) Infection and Immunity 62:1289-1297.



Study Design
P. gingivalis was cultured under standard anaerobic conditions.
After 8-10 days growth, the bacteria were harvested by
centrifugation.
Conditioned culture medium containing bacterial metabolic byproducts
was collected in the supernatant fraction
and bacteria were collected in the pellet fraction.
Bacterial cell pellets were washed, resuspended in buffer and
sonicated to break apart the bacteria forming a
soluble bacterial extract.
The soluble bacterial extract was fractionated by sequential
Centricon ultrafiltration to produce five
fractions-1) ? kDa, 2) 50-100 kDa, 3) 10-50 kDa, 4) 5-10 kDa, 5) ?
kDa.
Chicken periosteal osteogenesis (CPO) cultures was prepared from the
craniums of embryonic chickens and grown in culture under standard
conditions.

Loomer et al., (1994) Infection and Immunity 62:1289-1297.


Experiments
P. gingivalis spent culture medium and fractionated sonicated
extracts of whole P. gingivalis were added to osteogenic cultures in
various proportions ranging from 20 to 60% (vol/vol) .
Effects on CPO culture bone formation capacity was measured by the
effects on collagen synthesis and mineral uptake.
Collagen synthesis by osteogenic cultures was measured by the amount
of incorporation of 14C glycine into newly synthesized a -1 collagen
in 48 hrs by osteogenic cultures.
Mineral uptake by osteogenic cultures was measured by the amount of
total acid extractable calcium and phosphate incorporated by
osteogenic cultures in 48 hrs.

Loomer et al., (1994) Infection and Immunity 62:1289-1297.

Results
P. gingivalis conditioned medium significantly reduced both mineral
uptake and a-1collagen synthesis by osteogenic cultures.
The addition of 20% conditioned P. gingivalis culture medium to CPO
cultures reduced calcium accumulation by over 50%.
The addition of 25% P. gingivalis culture medium reduced phosphate
levels in CPO cultures by over 75%.

Whole bacterial sonicated extract fractions also significantly
reduced both mineral uptake and a-1 collagen synthesis.
All sonicated extract fractions reduced mineral uptake and collagen
synthesis but the greatest reduction was observed following treatment
with the ? kDa fraction which reduced collagen synthesis by over 90%
compared to controls (p?.001).
The < 5kDa fraction reduced calcium and phosphate accumulation below
the threshold level of detection.

Loomer et al., (1994) Infection and Immunity 62:1289-1297.

Conclusions
"Products derived from conditioned media or extracts of P.
gingivalis have the capacity to inhibit bone formation directly."
"The results obtained with the ? kDa fraction were unique in that
virtually complete inhibition of osteogenesis was observed at all
tested concentrations."
"The active compounds contained in this fraction were probably
neither protein nor intact LPS, as their molecular weights would be
too great. However, this fraction may contain high levels of organic
acids."
"Production of nonvolatile organic acids, for example, phenylacetic
acid and others, by P. gingivalis had been found to be exceedingly
high and likely has the potential to affect osteoprogenitor cell
differentiation profoundly."

Loomer et al., (1994) Infection and Immunity 62:1289-1297.

3 Classes of Non-Protein Bacterial Toxins
Volatile Sulfur Compounds (VSCs)
Hydrogen sulfide
Methyl mercaptan
Dimethyl sulfide
Dimethyl disulfide

Polyamines
Diaminobutane (Putrescine)
Diaminopentane (Cadaverine)
Spermidine

Short-Chain Carboxylic Acids (SSCAs)
Acetic acid
Propionic acid
Butyric acid


Production of Volatile Sulfur Compounds by Oral Bacteria
Volatile sulfur compounds (VSCs) together with volatile fatty acids
are largely responsible for oral malodor (halitosis).1
The two major VSCs are hydrogen sulfide (H2S) and methylmercaptan
(CH3SH) which account for about 90% of the total VSCs found in mouth
air.
H2S and CH3SH are thiols derived primarily from the breakdown of
protein substrates in saliva and dental deposits by oral bacteria.2
H2S is derived from the breakdown of the amino acid L-cysteine.
CH3SH is derived from the breakdown of L-methionine.

The levels of H2S and CH3SH in periodontal pockets and mouth air
have been found to correlate with the
severity of periodontal disease.
1De Boever et al., (1994) J. Clin. Dent. 4:114-119. 2Persson et al.,
(1992) Oral Microbiol.
Immunol. 7:378-379.

The Bacterial Enzyme L-Cysteine Desulfhydrase Catalyzes the Breakdown
of L-Cysteine into Hydrogen Sulfide and
Ammonium

The Bacterial Enzyme L-Methionine-?-Lyase Catalyzes the Breakdown of
the Amino Acid L-Methionine into Methyl
Mercaptan

Sources and Levels of VSCs
Anaerobic bacteria in infected periodontal pockets produce H2S and
CH3SH in toxic amounts. *
H2S levels have been found to be as high as 2 mM in infected
gingival pockets.*
75 different species of oral bacteria have been identified that can
degrade L-cysteine and cysteine containing peptides and proteins into
H2S.*
21 different of species of oral bacteria have been identified that
can degrade methionine and methionine containing peptides and
proteins into CH3SH.*

*Persson,S. (1992). Hydrogen sulfide and methyl mercaptan in
periodontal pockets. Oral Microbiol. Immunol. 7:378-379.
Mechanisms of Hydrogen Sulfide Toxicity
H2S binds to metal ion cofactors of proteins inhibiting their
activities.
H2S binds the Fe3+ in the heme moiety of the mitochondrial enzyme
cytochrome a3 oxidase similar to hydrogen cyanide inactivating the
enzyme and blocking the terminal step in the electron transport
system. 1
H2S binds to Zn2+ in carbonic anhydrous replacing the bound hydroxyl
necessary for the interconversion of CO2 and water to bicarbonate.1

1Beauchamp et al., (1984) CRC Crit. Rev. Toxicol. 13:25-97.
Mechanisms of Hydrogen Sulfide Toxicity (cont.)
H2S reduces essential disulfide bonds of cellular proteins leading
to loss of protein tertiary structure and activity.
H2S directly inhibits monoamine oxidase and acetylcholinesterase
resulting in changes in catecholamine and acetylcholine
neurotransmitter content .2
H2S inhibits the Na+/K+-ATPase resulting in disturbances in ion
balance.2
H2S reduction of compliment C3bi inhibits binding of the protein to
the compliment receptor on PMN leukocytes blocking the ability of
these white blood cells to phagocytize and kill bacteria.3
2Reiffenstein et al., (1992) Annu. Rev. Pharmacol. Toxicol. 32:109-
134. 3Granlund-Edstedt et al., (1993) J. Periodont. Res. 28:346-
353.

Toxic Effects of H2S and CH3SH on Oral Tissues
Human Gingival Fibroblasts1
Solubilization of collagen
Increased permeability of the oral mucosa to ions, prostaglandins,
and bacterial endotoxins
Inhibition of protein synthesis
Decreased synthesis and increased degradation of collagen

Polymorphonuclear Leukocytes (PMNs)2
Inhibition of killing of bacteria by PMN due to inhibition of
complement binding to bacteria

Bone Formation 3
Decreased collagen production
Decreased phosphate and calcium deposition (mineralization)
1Johnson et al., (1992). J. Periodont. Res. 27:553-561 2Granlund-
Edstedt et al., (1993). J. Periodont. Res. 28:346-353. 3Loomer et
al., (1994). Infect. Immun. 62:1289-1297.


Effects of H2S and CH3SH on Membrane Permeability
Hydrogen sulfide (H2S) and methyl mercaptan (CH3SH) act as
permeation agents.1
Low concentrations of H2S and CH3SH increase the permeability of the
gingival epithelial tissue and oral mucosa.2
Alterations in the integrity of the mucosal barrier results from
reaction of H2S and CH3SH with cellular proteoglycans and other
matrix components such as collagen resulting in loss of tertiary
structure and increased degradation.
Increased permeability allows bacterial antigens to penetrate the
gingival epithelium and reach the underlying connective tissue. 2
Bacterial antigens initiate an inflammatory reaction which increases
the rate of connective tissue breakdown leading to gingivitis and
periodontal disease. 1,2
1Rizzo (1970) J. Periodontol. 41:210-212. 2Ng and Tonzetich (1984)
J. Dent. Res. 63:994-997.

Toxicity of Hydrogen Sulfide
Hydrogen sulfide (H2S) is a highly toxic, colorless gas that is
emitted from natural sources and during man made activities.
H2S is an both environmental and industrial pollutant, being
produced by many common bacteria and in over 70 industrial
processes.
H2S is found in high concentrations in many deposits of petroleum
and natural gas. Oil, natural gas and petrochemical industrial
accidents account for most of the documented human H2S exposures.
Human exposure to H2S has been reported secondary to a variety of
industrial accidents involving acute, high-dose exposures and long-
term chronic exposures.
The primary effects of exposure to H2S depend on the concentration
in the inhaled air and include impaired visual acuity, conjunctival
and nasal irritation, olfactory paralysis, pulmonary edema and
nervous stimulation of respiration, followed by respiratory paralysis
and death.
Reiffenstein, Hulbert and Roth (1992) Toxicology of hydrogen
sulfide.
Annu. Rev. Pharmacol. Toxicol. 32:109-134.

Human Physiologic Responses to H2S Exposure
Adapted from Reiffenstein et al., (1992) Toxicology of Hydrogen
Sulfide. Ann. Rev. Pharmacol.
Toxicol. 32:109-134.

The hydrogen sulfide produced by anaerobic bacteria is the same,
chemically speaking, as the hydrogen sulfide
produced as a waste product in over 70 different industrial
processes.
The toxic properties of the compound does not differ depending upon
the source of its production.

Neurotoxic Effects of Hydrogen Sulfide
Effects of repeated exposures of hydrogen sulfide on rat hippocampal
EEG. Skrajny et al., (1996). Toxicol. Lett. 84:43-53.
Alteration of the morphology and neurochemistry of the developing
mammalian nervous system by hydrogen sulfphide. Roth et al., (1995).
Clin. Exp. Pharmacol. Physiol. 22:379-380.
Hydrogen sulfide and reduced-sulfur gases adversely affect
neurophysiological functions. Kilburn and Warshaw (1995). Toxicol.
Ind. Health 11:185-197.
Sulfide-induced perturbations of the neuronal mechanisms controlling
breathing in rats. Greer et al., (1995). J. Appl. Physiol. 78:433-
440.
The actions of hydrogen sulfie on dorsal raphe serotonergic neurons
in vitro. Kombian et. al., (1993). J. Neurophysiol. 70:81-96.
Low concentrations of hydrogen sulfide alter monoamine levels in the
developing rat central nervous system. Skrajny et al., (1992). Can.
J. Physiol. Pharmacol. 70:1515-1518.
Chronic exposure to low concentrations of hydrogen sulfide produces
abnormal growth in developing cerebellar Purkinje cells. Hannah and
Roth (1991). Neurosci. Lett. 28:225-228.
Hydrogen sulfide exposure alters the amino acid content in
developing rat CNS. Hannah et al. (1989). Neurosci. Lett. 8:323-327.
Monoamine oxidase inhibition as a sequel of hydrogen sulfide
intoxication: increases in brain catecholamine and 5-
hydroxytryptamine levels. Warenycia et al., (1989). Arch. Toxicol.
63:131-136.

Toxic Effects of Hydrogen Sulfide and Methylmercaptan
Modulation of human gingival fibroblast cell metabolism by methyl
mercaptan. Johnson et al. (1992). J. Periodontal Res. 27:476-483.
Effect of volatile thiol compounds on protein metabolism by human
gingival fibroblasts. Johson et. al., (1992). J. Periodont. Res.
27:553-561.
The effect of methanethiol and methionine toxicity on the activities
of cytochrome c oxidase and enzymes involved in protection from
peroxidative damage. Finkelstein and Benevenga (1986). J. Nutr.
116:204-215.
Effects of methanethiol on erythrocyte membrane stabilization and on
Na+,K+-adenosine triphosphatase: relevance to hepatic coma. Ahmed et
al., (1984). J. Pharmacol. Exp. Ther. 228:103.
Acute and subchronic toxicity studies of rats exposed to vapors of
methyl mercaptan and other reduced-sulfur compounds. Transy et al.,
(1981). J. Toxicol. Environ. Health 8:71-88.
Cytotoxic effects of hydrogen sulfide on pulmonary alveolar
macrophages in rats. Khann et al., (1991). J. Toxicol. Environ.
Health 33:57-64.
Exposure to low levels of hydrogen sulfide elevates circulating
glucose in maternal rats. Hayden et al., (1990). J. Toxicol. Environ.
Health 31:45-52.
Toxicology of hydrogen sulfide. Reiffenstein et al., (1992). Ann.
Rev. Pharmacol. Toxicol. 32:109.
Hydrogen sulfide: a bacterial toxin in ulcerative colitis? Pitcher
and Cummings (1996). Gut 39:1-4.

Effect of Toxins on Photolabeling of a Mixture of Purified Mammalian
Nucleotide Binding Proteins

Hydrogen Sulfide & Methylmercaptan Inhibit [32P]N3ATP Interactions
with Purified Mammalian Enzymes

3 Classes of Non-Protein Bacterial Toxins
Volatile Sulfur Compounds (VSCs)
Hydrogen sulfide
Methyl mercaptan
Dimethyl sulfide
Dimethyl disulfide

Polyamines
Diaminobutane (Putrescine)
Diaminopentane (Cadaverine)
Spermidine

Short-Chain Carboxylic Acids (SSCAs)
Acetic acid
Propionic acid
Butyric acid


Formation of Polyamines by Oral Bacteria
Polyamines are intracellular organic cations which help regulate a
number of important biological activities in both prokaryotic and
eukaryotic cells. These include DNA replication, transcription and
translation as well as the activities of several types of protein
kinases.
High polyamine concentrations in biological fluids such as GCF can
occur as a result of cell lysis or cell proliferation, both of which
occur to host cells and bacteria at sites of bacterial infection
Cadaverine and putrescine are both common bacterial degradation
products
Cadaverine is produced by the decarboxylation of the amino acid
lysine
Putrescine in produced by the decarboxylation of the amino acid
ornithine and by the transamination of the amino acid arginine

Spermidine is produced from putrescine and aminopropyl residues from
decarboxylated S-adenosylmethionine by the enzyme spermidine
synthase.
Hayes and Hyatt (1974) Arch. Oral. Biol. 19:361-369. Masui and
Kirimura (1987) Shigaku 75:117-136. Goldberg et al., (1994) J. Dent.
Res. 73:1168-1172.
Relationship Between Polyamines and Periodontitis
Putrescine and cadaverine concentrations are positively correlated
with pocket probing depth. Since host cells contain very little
putrescine and no cadaverine, subgingival bacteria were presumed to
be responsible.1
Spermidine concentrations are significantly elevated (3-fold) in
inflammed gingival sites (Gingival Index=2). 1
GCF polyamine concentrations were highly variable between sites with
similar probing depths and gingival index scores. Levels often varied
several-fold from site to site within the same subject. 1
Polyamine concentrations in GCF positively correlate with the level
of spirochetes in moderately inflamed periodontits sites.2
Cadaverine levels in human saliva have been shown to be positively
correlated with the Plaque Index (PI), Gingival Index (GI), probing
depth, BANA scores but not with VSC levels.3
1 Walters (1987) J. Periodont. Res. 22:522-523. 2Walters et al.,
(1989) Arch. Oral Biol. 34:373-375. 3Goldberg et al., (1994). J.
Dent. Res. 73:1168-1172.

Relationship Between Polyamines and Periodontitis
Putrescine and cadaverine concentrations are positively correlated
with pocket probing depth. Since host cells contain very little
putrescine and no cadaverine, subgingival bacteria were presumed to
be responsible.1
Spermidine concentrations are significantly elevated (3-fold) in
inflammed gingival sites (Gingival Index=2). 1
GCF polyamine concentrations were highly variable between sites with
similar probing depths and gingival index scores. Levels often varied
several-fold from site to site within the same subject. 1
Polyamine concentrations in GCF positively correlate with the level
of spirochetes in moderately inflamed periodontits sites.2
Cadaverine levels in human saliva have been shown to be positively
correlated with the Plaque Index (PI), Gingival Index (GI), probing
depth, BANA scores but not with VSC levels.3


1 Walters (1987) J. Periodont. Res. 22:522-523. 2Walters et al.,
(1989) Arch. Oral Biol. 34:373-375. 3Goldberg et al., (1994). J.
Dent. Res. 73:1168-1172.
Polyamines Produced by Oral Microorganisms

Cadaverine is Produced from Lysine by the Action of the Bacterial
Enzyme Lysine Decarboxylase

Putrescine is Produced from Ornithine by the Action of the Bacterial
Enzyme Ornithine Decarboxylase

Polyamine Concentrations Measured in Human GCF and Saliva of Persons
with Periodontitis
1Walters (1987). Polyamine analysis of human gingival crevicular
fluid. J. Periodont. Res. 22:522-523. 2Lamster et al., (1987). The
polyamines putrescine, spermidine and spermine in human gingival
crevicular fluid. Archs. Oral Biol. 32:329-333. 3Goldberg et al.,
(1994). Cadaverine as a putative component of oral malodor. J. Dent.
Res. 73:1168-1172.

ND=Not determined


Polyamines in Gingival Crevicular Fluid
Polyamines found in gingival fluid inhibit chemotaxis by human
polymorphonuclear leukocytes in vitro. Walters et al., (1995). J.
Periodontol. 66:274-278.
Polyamines found in gingival fluid enhance the secretory and
oxidative function of human polymorphonuclear leukocytes in vitro.
Walters and Chapman (1995). J. Periodontal. Res. 30:167.
Cadaverine as a putative component of oral malodor. Goldberg et al.,
(1994). J. Dent. Res. 73:1168-1172.
Polyamine analysis of infected root canal contents related to
clinical symptoms. Maita and Horiuchi (1990). Endod. Dent. Traumatol.
6:213-217.
Relationship of human gingival crevicular fluid polyamine
concentration to the percentage of spirochaetes in subgingival dental
plaque. Walters et al., (1989). Arch. Oral Biol. 34:373-375.
Studies on polyamine of gingival tissues in marginal periodontitis.
Shinohara et al., (1988). Nippon Shishubyo Gakki Kaishi 30:142-147.
The polyamines putrescine, spermidine and spermine in human gingival
crevicular fluid. Lamster et al., (1987). Arch. Oral Biol. 32:329-
333.
Polyamine analysis of human gingival crevicular fluid. Walters
(1987). J. Periodontal. Res. 22:522-523.
Production of putrescine from arginine by oral microorganisms. Masui
and Kirimura (1987). Shigaku 75:117-136.

Polyamine Analysis of Infected Root Canal Contents Related to
Clinical Symptoms. Maita & Horiuch (1990). Endod. Dent. Traumatol.
6:213-217.
Contents of infected root canals were collected by mechanical and
chemical debridement.
Intracanal polyamines were identified & quantified by HPLC.
The amounts of polyamines were significantly greater in teeth with
spontaneous pain, swelling and putrescent odor, with exudate and with
percussion pain than in teeth without.
Putrescine amounts were greater in teeth with spontaneous pain and
percussion pain than in teeth without clinical sign.
Cadaverine amounts were greater in teeth with gingival fistulae than
from those without.


3 Classes of Non-Protein Bacterial Toxins
Volatile Sulfur Compounds (VSCs)
Hydrogen sulfide
Methyl mercaptan
Dimethyl sulfide
Dimethyl disulfide

Polyamines
Diaminobutane (Putrescine)
Diaminopentane (Cadaverine)
Spermidine

Short-Chain Carboxylic Acids (SSCAs)
Acetic acid
Propionic acid
Butyric acid


Short-Chain Carboxylic Acids (SSCAs) Produced by Oral Microorganisms
Adapted from Fig. 3, Niederman et al., (1997). Crit. Rev. Oral Biol.
Med. 8:269-290.

Production of Short-Chain Carboxylic Acids by Oral Microorganisms
Short-chain carboxylic acid concentration in human gingival
crevicular fluid. Niederman et al., (1997). J. Dent. Res. 76:575-
579.
Short-chain carboxylic acid-stimulated, PMN-mediated gingival
inflammation. Niederman et al., (1997). Crit. Rev. Oral Biol. Med.
8:269-290.
Bacterial metabolite mediated differential human PMN gene
expression. Swartout and Niederman (1997). J. Periodont. Res. 32:196-
199.
The relationship of gingival crevicular fluid short chain carboxylic
acid concentration to gingival inflammation. Niederman et al.,
(1996). J. Clin. Periodontol. 21:743-749.
Volatile fatty acids, metabolic by-products of periodontopathic
bacteria, inhibit lymphocyte proliferation and cytokine production.
Kurita-Ochiai et al., (1995). J. Dent. Res. 74:1367-1373.
Short chain fatty acids produced by anaerobic bacteria inhibit
adhesion and proliferation of periodontal ligament fibroblasts.
Eftimiadi et al., (1993). Minerva Stomatol. 42:481-485.
Divergent effect of the anaerobic bacteria by-product butyric acid
on the immune response: suppression of T-lymphocyte proliferation and
stimulation of interleukin-1 beta production. Eftimaidi et al.,
(1991). Oral Microbiol. Immunol. 6:17-23.
Anaerobes and short-chain fatty acids in crevicular fluid from
adults with chronic periodontitis. Courtois et al., (1989). Bull
Group Int. Rech. Sci. Stomatol. Odontol. 32:19-22.
Butyrate: a cytotoxin for Vero cells produced by Bacteriodes
gingivalis and Bacteriodes asaccharolyticus. Touw et al., (1982).
Ant. Van Leeuwenhoek 48:315-325.

Common Metabolic Pathways for Production of Short-Chain Carboxylic
Acids by Oral Bacteria
Adapted from Fig. 3, Niederman et al., (1997). Crit. Rev. Oral Biol.
Med. 8:269-290.
Toxic Effects of Short-Chain Carboxylic Acids
Stimulate a gingival inflammatory response and inflammatory cytokine
release when applied to healthy human gingiva.
Inhibit proliferation of gingival epithelial and endothelial cells.
Inhibit neutrophil chemotaxis, phagocytosis, killing and
degranulation.
Alter neutrophil gene transcription, translation, and protein
expression.
Stimulate neutrophil cytokine production and release.
Inhibit neutrophil apoptosis prolonging the inflammatory reaction.
Inhibit T- and B-lymphocyte proliferation and cytokine production.


Niederman et al., (1997). Short-chain carboxylic acid stimulated
PMN -mediated gingival inflammation. Crit. Rev. Oral Biol. Med.
8:269-290.
Concentrations of Short-Chain Carboxylic Acids Measured in GCF of
Patients with Periodontitis
* = 3 months after scaling and root planing, N.D. = Not Detectable

Niederman et al., (1997). J. Dent. Res. 76:575-579.

SCCA concentration positively correlated with clinical measures of
disease severity( e.g. pocket depth, attachment level) and gingival
inflammation (subgingivial temperature, % of sites bleeding when
probed) but not with the total or individual numbers of any putative
periodontal pathogens.
Production & Toxicity of Short-Chain Carboxylic Acids
Short chain carboxylic acids decrease human gingival keratinocyte
proliferation and increase apoptosis and necrosis. Sorkin and
Niederman (1998). J. Clin. Periodontol. 25:311-315.
Short-chain carboxylic acid concentration in human gingival
crevicular fluid. Niederman et al., (1997). J. Dent. Res. 76:575-
579.
Short-chain carboxylic-acid-stimulated, PMN-mediated gingival
inflammation. Niederman et al., (1997). Crit. Rev. Oral Biol. Med.
8:269-290.
Bacterial metabolites sodium butyrate and propionate inhibit
epithelial cell growth in vitro. Pollanen et al., (1997). J.
Periodontal. Res. 32:326-334.
Bacterial metabolite mediated differential human PMN gene
expression. Swartout and Niederman (1997). J. Periodont. Res. 32:196-
199.
The relationship of gingival crevicular fluid short chain carboxylic
acid concentration to gingival inflammation. Niederman et al.,
(1996). J. Clin. Periodontol. 23:743-749.
Effects of the Prevotella intermedia culture filtrate and short-
chain fatty acids on human polymorphonuclear neutrophil functions.
Touyama et al., (1995). Kansenshogaku Zasshi 69:1348-1355.
Volatile fatty acids, metabolic by-products of periodontopathic
bacteria, inhibit lymphocyte proliferation and cytokine production.
Kurita-Ochiai et al., (1995). J. Dent. Res. 74:1367-1373.

Production & Toxicity of Short-Chain Carboxylic Acids
Short chain fatty acids produced by anaerobic bacteria inhibit
adhesion and proliferation of periodontal ligament fibroblasts.
Eftimiadi et al., (1993). Minerva Stomatol. 42:481-485.
Divergent effect of the anaerobic bacteria by-product butyric acid
on the immune response: suppression of T-lymphocyte proliferation and
stimulation of interleukin-1 beta production. Eftimaidi et al.,
(1991). Oral Microbiol. Immunol. 6:17-23.
Anaerobes and short-chain fatty acids in crevicular fluid from
adults with chronic periodontitis. Courtois et al., (1989). Bull
Group Int. Rech. Sci. Stomatol. Odontol. 32:19-22.
Butyrate: a cytotoxin for Vero cells produced by Bacteriodes
gingivalis and Bacteriodes asaccharolyticus. Touw et al., (1982).
Ant. Van Leeuwenhoek 48:315-325.
High carboxylic acid level in the gingival crevicular fluid (GCF) of
the patients with advanced periodontal disease. Ohwaki (1988). Nippon
Shishubyo Gakkai Kaishi 30:985-995.
Isotachophoretic analysis of short-chain carboxylic acids produced
by Candida albicans. Samaranayake et al., (1982). Microbios. 35:91-
98.
Production of phenylacetic acid by strains of Bacteroides
asaccharolyticus and Bacteriodes gingivalis (sp. nov.) Kaczmarek and
Coykendall (1980). J. Clin. Microbiol. 12:288-290.

Short-Chain Carboxylic Acids Inhibit [32P]N3ATP Interactions with
Purified Mammalian Enzymes

Conclusions
High (millimolar) levels of non-protein bacterial toxins are present
at sites of active infection in the mouth.
This includes both periodontal and endodontic infections.
These bacterial toxins, at levels measured at sites of active
infection, can inhibit the activity of a number of vital mammalian
enzymes in a dose related manner.
The technique of nucleotide photoaffinity labeling with purified
mammalian enzymes can be used to detect the presence of these toxins
in gingival crevicular fluid (see the GCF Test) or extracts prepared
from non-vital teeth.
Toxicity assays based on reaction of these bacterial toxins with
colorless chemical reagents to produce colored reaction products can
be used to detect and quantify the levels of these toxins (see the
TOPAS).


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