J Periodontol.

 1985 Mar;56(3):139-47.

Lactate dehydrogenase, beta-glucuronidase and arylsulfatase

activity in gingival crevicular fluid associated with
experimental gingivitis in man.
Lamster IB, Vogel RI, Hartley LJ, DeGeorge CA, Gordon JM.
Abstract
Experimental gingivitis provides a useful model for studying the initiation of periodontal disease in
man. This study evaluated over a 4-week period the Plaque Index (PLI), Gingival Bleeding Time Index
(GBTI), and gingival crevicular fluid (GCF) for resting and flow volume as well as the concentration
and total activity of three enzymes in the GCF (lactate dehydrogenase--LDH, beta-glucuronidase--BG
and arylsulfatase--AS) from the maxillary right quadrant of eight subjects with healthy gingiva. After
rising sharply during the 1st week, the PLI continued to increase during the 2nd week but remained
constant during the 3rd and 4th weeks. The GBTI, and the resting and flow GCF volumes, increased
steadily throughout the study. LDH concentration in GCF varied minimally during the experiment,
while total LDH activity rose slightly over the 4-week period. BG concentration and total activity in
GCF rose steadily from baseline to the 3rd week and then either fell or leveled off during the 4th
week. AS concentration in GCF rose from baseline to the 2nd or 3rd week and then fell. AS total
activity in GCF rose from baseline to the 2nd week and then remained constant. These data suggest
that while clinical signs of inflammation increased over the 4 weeks of the experiment, a homeostatic
mechanism in the crevicular environment may control ground substance-degrading enzyme activity
during experimental gingivitis in man.

Review

Carlo Cafiero and Sergio Matarasso .Predictive, preventive, personalised and participatory

periodontology: ‘the 5Ps age’ has already started. EPMA
Journal 2013, 4:16 doi:10.1186/1878-5085-4-16

 *Corresponding author: Carlo Cafiero c.cafiero@unina.it

Author Affiliations

University of Naples “FEDERICO II”, Naples 80131, Italy

For all author emails, please log on.

The electronic version of this article is the complete one and can be found online at:http://www.epmajournal.com/content/4/1/16

Received: 19 March 2013

Accepted: 6 May 2013

Published
: 14 June 2013

© 2013 Cafiero and Matarasso; licensee BioMed Central Ltd. 

This is an Open Access article distributed under the terms of the Creative Commons Attribution License

(http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium,

provided the original work is properly cited.

Abstract
An impressive progress in dentistry has been recorded in the last decades. In order to reconsider guidelines in dentistry, it is required
to introduce new concepts of personalised patient treatments: the wave of predictive, preventive and personalised medicine is
rapidly incoming in dentistry. Worldwide dentists have to make a big cultural effort in changing the actual ‘reactive’ therapeutic
point of view, belonging to the last century, into a futuristic ‘predictive’ one. The first cause of tooth loss in industrialised world is
periodontitis, a Gram-negative anaerobic infection whose pathogenesis is genetically determined and characterised by complex
immune reactions. Chairside diagnostic tests based on saliva, gingival crevicular fluid and cell sampling are going to be routinely
used by periodontists for a new approach to the diagnosis, monitoring, prognosis and management of periodontal patients. The
futuristic ‘5Ps’ (predictive, preventive, personalised and participatory periodontology) focuses on early integrated diagnosis
(genetic, microbiology, host-derived biomarker detection) and on the active role of the patient in which networked patients will shift
from being mere passengers to responsible drivers of their health. In this paper, we intend to propose five diagnostic levels (high-
tech diagnostic tools, genetic susceptibility, bacterial infection, host response factors and tissue breakdown-derived products) to be
evaluated with the intention to obtain a clear picture of the vulnerability of a single individual to periodontitis in order to organise
patient stratification in different categories of risk. Lab-on-a-chip (LOC) technology may soon become an important part of efforts
to improve worldwide periodontal health in developed nations as well as in the underserved communities, resource-poor areas and
poor countries. The use of LOC devices for periodontal inspection will allow patients to be screened for periodontal diseases in
settings other than the periodontist practice, such as at general practitioners, general dentists or dental hygienists. Personalised
therapy tailored with respect to the particular medical reality of the specific stratified patient will be the ultimate target to be realised
by the 5Ps approach. A long distance has to be covered to reach the above targets, but the pathway has already been clearly
outlined.

Keywords: 
Predictive periodontology; Preventive periodontology; Personalised periodontology; Participatory periodontology; Lab-on-a-chip;
Gas chromatographs; Cone beam computed tomography; Host-derived diagnostic markers; Saliva; Gingival crevicular fluid

Review
Introduction
Not too many years ago, the most frequent therapy in dentistry was tooth extraction: the teeth were pulled out and rapidly
substituted by the application of a fixed or mobile prosthetic appliance. At that time, due to the weakness of the background in
dental researches, just a small number of dentists were able to perform dental therapies on a specialised level. In the majority of
cases, advanced therapies were generally considered not more than pioneeristic attempts. An impressive progress in dentistry has
been recorded in the last decades. Synergic efforts (understanding of biological phenomena, new biomaterials, sophisticated surgical
techniques, high tech in diagnostic tools, etc.) have carried dentistry away from the middle-aged situation described above
(Figure 1), but this is not enough. In order to reconsider guidelines in dentistry, it is required to introduce new concepts of
personalised patient treatments. On account of this, the current paper follows the recommendations of the recently published ‘White
Paper’ of the European Association for Predictive, Preventive and Personalised Medicine (EPMA) [1,2]. The wave of predictive,
preventive and personalised medicine is quickly incoming in dentistry. With regard to this, the mission of a specialised EPMA
dental section will be to aid worldwide dentists make a big cultural effort in changing the actual ‘reactive’ therapeutic point of view,
belonging to the last century, into a futuristic ‘predictive’ one (Figure 2). Enhancement in dental knowledge revealed genetic,
microbiological and immunological mechanisms at the base of the most common dental diseases.

Figure 1. Advances in biological and technical research are clearly outlining the pathway for the future

of dentistry.
 Figure 2. EPMA is present in 44 countries worldwide. The EPMA Department for Predictive, Preventive

and Personalised Dentistry is going to make a big cultural effort in changing worldwide dentists' therapeutic point of view from a

reactive to a predictive one. (Adapted from [1]).

The first cause of tooth loss in industrialised world is periodontitis that strikes prevalently people older than 40 years of age. In
consequence of this, the prevention of periodontitis is of capital importance to general and oral health since the European population
is becoming progressively older (Figure 3). Periodontitis is a Gram-negative anaerobic infection whose pathogenesis is genetically
determined and characterised by complex immune reactions to bacterial burden. It constitutes a very interesting model of chronic
oral pathology, characterised by activity phases, related to many branches of medical researches such as genetic, microbiology and
immunology. On account of this, chairside diagnostic tests based on saliva, gingival crevicular fluid and cell sampling are going to
be routinely used by periodontists for a novel approach to the diagnosis, monitoring, prognosis and management of periodontal
patients. As will be discussed later, genetic tests as well as the use of microbial analysis and the detection of biomarkers derived
from host response will contribute to improve periodontal health. Predictive, preventive, personalised and participatory
periodontology, the ‘5Ps’, represents with no doubt the future of the profession of periodontology. A predictive approach due to the
use of high-tech diagnostic tools will give us the possibility to detect patients at risk and to effect early diagnosis of periodontitis
when it is easier to treat successfully. It will be organised as a personalised prevention, based upon the genetic and microbiological
status of a single patient as well as personalised therapy tailored with respect to the particular medical reality of the specific patient.
Finally, the active role of the patient will be emphasised through the introduction of participatory periodontology, a concept in
which networked patients will shift from being mere passengers to responsible drivers of their health.

Figure 3. Since the European population is becoming progressively older, the prevention of

periodontitis is of capital importance. Well-organised screenings have to be performed in order to organise targeted prevention

and cost-effective healthcare.

The aim of this paper is to synthetically report actual information on the genetics, microbiology and immunology of periodontal
disease related to biomarkers that can aid to have early diagnosis. Further biomarkers, coming out in the early destruction of
periodontal tissues, will be equally reported and discussed. The present article is directed not only to dental operators (such as
general dentists, periodontists, dental hygienists) but also to medical doctors in order to enlarge the discussion group and to share
our experiences and ideas with as much colleagues as possible. Considering the large number of professionals we intend to
approach with the present paper, it seems clear that we have to discuss some basic topics about periodontal disease before
introducing the specific periodontal biomarkers field.

Periodontal unit as a multi-functional complex

The periodontium is defined as an anatomic and functional complex which constitutes the supporting tissue of the teeth. Each of the
periodontal components has its very specialised function. Periodontal tissues are distinct in the (1) gingiva and (2) deep
periodontium (periodontal ligament, cementum and alveolar bone).

Gingiva
The gingiva is a coral pink tissue consisting of an epithelial layer and an underlying connective tissue. It is differentiated into
marginal (free) gingiva and attached gingiva. The free gingiva extends at the vestibular and lingual/palatal aspects of the teeth and
in the interdental space which constitutes the peak of interdental papillae. On the vestibular and lingual sides, the free gingiva
extends from the gingival margin to the free gingival groove which marks the edge with the attached gingiva. The attached gingiva
extends in the apical direction to the mucogingival junction where it becomes continuous with the alveolar mucosa [3] (Figure 4).

Figure 4. Marginal (free) gingiva, attached gingiva and alveolar mucosa.

Deep periodontium
The deep periodontium is composed of the periodontal ligament, cementum and alveolar bone (Figure 5).
 Figure 5. Deep periodontium. The main function of the deep periodontium is to support the teeth in their

sockets and to act as a sensory receptor necessary for the proper positioning of the jaws and the proper pressure to be exercised

during mastication.
Periodontal ligament
The periodontal ligament (alveolo-dental ligament) is a specialised connective tissue situated between the cementum covering the
root of the tooth and the bone forming the socket wall. The extremities of collagen fibre bundles are embedded in the cementum
(Sharpey's fibres) on one side and in the alveolar bone on the other side. It ranges in width from 0.15 to 0.38 mm.

Cementum
The cementum is the hard, avascular connective tissue covering the roots of the teeth that serves primarily to attach the principal
periodontal ligament fibres. There are two principal varieties of the cementum classified on the basis of the presence or absence of
cells: acellular extrinsic fibre cementum (primary cementum or acellular cementum) and cellular intrinsic fibre cementum
(secondary cementum or cellular cementum). The acellular extrinsic fibre cementum extends from the cervical half to two thirds of
the root. The high number of Sharpey's fibres inserting in it shows its fundamental function in tooth attachment. The cellular
intrinsic fibre cementum is distributed along the apical third or half of the root and in furcation areas. It represents a reparative
tissue.

Alveolar bone
The mineralised bone is made up of lamellae (lamellar bone). It includes two types of bone tissue, the bone of the alveolar process
and the alveolar bone lining the socket referred to as the alveolar bone proper or ‘bundle bone’ that consists of intrinsic fibre
bundles running parallel to the socket. Embedded within this bundle bone and perpendicular to its surface are Sharpey's fibres. The
alveolar bone is a clear example of a structure-function relationship because it increases in conjunction with the development of the
teeth and it is partially lost in the absence of a tooth. In conclusion, the principal function of deep periodontal tissues is to support
the teeth in their sockets. In addition, periodontal tissues act as a sensory receptor necessary for the proper positioning of the jaws
and the proper pressure to be exercised during mastication. The peripheral feedback coming from the periodontal ligament gives
signals to the muscles, ear and temporomandibular joints about the quality of the food present under the teeth and, as a consequence,
the information for the fine-tuning of masticatory pressure.

Periodontal diseases: the real infections of the oral cavity

Periodontal diseases are a cluster of inflammatory bacterial plaque-induced pathologies. At the end of the 1990s, a classification
system for periodontal diseases was proposed, and it is currently used worldwide (see below) [4]:
1. Gingivitis

2. Chronic periodontitis

3. Aggressive periodontitis

4. Periodontitis as a manifestation of systemic disease

5. Necrotising ulcerative gingivitis/periodontitis

6. Abscesses of the periodontium

7. Combined periodontic-endodontic lesions

The most common periodontal diseases are gingivitis and periodontitis whose primary characteristics are synthetically reported
below.
Gingivitis
Gingivitis is an inflammation of the periodontal marginal tissue (gingiva) in response to bacterial biofilms (bacterial plaque)
adherent to tooth surfaces [5]. It is characterised by redness, bleeding, volume augmentation and diffuse pain (Figures 6, 7, 8,
and 9). Gingivitis is a non-destructive periodontal disease in which no deep connective tissue destruction or bone resorption is
detectable. In the presence of periodontal treatment, a complete resolution of the disease andrestitutio ad integrum is to be expected.

Figure 6. Upper jaw acute gingivitis in a non-smoker 26-year-old male patient. Abundant plaque deposit

is visible on the surfaces of the teeth.

Figure 7. Lower jaw of the same patient. Calculus covers the entire surfaces of the teeth.

Figure 8. Upper jaw. Complete resolution of acute gingivitis andrestitutio ad integrum.

Figure 9. Lower jaw. The patient's compliance is of capital importance for a long-term result.
Periodontitis
Periodontitis is a destructive pathology caused by Gram-negative facultative anaerobes affecting periodontal tissues (gingiva,
cementum, periodontal ligament, alveolar bone). It causes periodontal breakdown (connective attachment loss, bone resorption and
formation of periodontal pockets) as result of a complex bacteria-host response in genetically oriented patients (Figure 10).
Periodontitis is characterised by a cyclic progression in which a recurrent active phase (periodontal breakdown) is followed by a
quiescence phase. The natural history of the disease determines progressive periodontal destruction, tooth mobility and migration.
This situation can lead to tooth loss and sometimes can render the patient edentulous. Therapy generally stops its progression and in
some cases (regenerative surgery) can lead to restitutio ad integrum. Periodontitis is classified into chronic and aggressive forms.

Figure 10. Periodontitis is a result of a complex bacteria-host response in genetically oriented

patients. Co-factors such as diabetes and cigarette smoking are strongly associated with the aggravation of periodontitis.
Chronic periodontitis
Chronic periodontitis (CP) affects up of 50% of the global population, especially older patients, but may occur in children too. In
most cases, the rate of progression of chronic periodontitis is slow, and the amount of periodontal tissue destruction is generally
commensurate with sub-gingival calculus and plaque amounts. CP is classified as localised when <30% of sites are affected
andgeneralised when this level is exceeded (Figures 11 and 12).

Figure 11. Generalised chronic periodontitis. The amount of periodontal tissue destruction commensurate

with sub-gingival calculus and plaque amounts, diffuse pathological probing depth, mobility and migration are the main

characteristics of this pathology.

Figure 12. Full-mouth series periapical X-rays show diffuse horizontal bone loss.
Aggressive periodontitis
Aggressive periodontitis (AP) is less common than the chronic form. In the primary dentition of 5–11-year-olds, the frequency
ranges from 0.9% to 1.5% of subjects [6-8], and in the permanent dentition of 12–20-year-olds, the frequency ranges from 0.1% to
0.2% in Caucasian populations. AP generally affects younger patients causing rapid loss of attachment and bone destruction. The
severity of periodontal tissue destruction is conflicting with the scarce amounts of microbial deposits. The reason of this destruction
is the presence of elevated proportions of aggressive Gram-negative bacteria (Aggregatibacter
actinomycetemcomitans and Porphyromonas gingivalis), the phagocyte abnormalities and the hyperresponsive macrophage
phenotype (elevated secretion of prostaglandin E2 (PGE2) and interleukin-1 (IL-1)) in response to bacterial lipopolysaccharides
(LPSs). Aggressive periodontitis has been sub-classified into localised and generalised forms[9,10].
Localised aggressive periodontitis
Localised aggressive periodontitis (LAP) presents a circumpubertal onset. The first molar/incisor presents with interproximal
attachment loss on at least two permanent teeth, one of which is the first molar, and involving no more than two teeth other than the
first molars and incisors. Serum antibody response to infecting agents was detected in high quantity.

Generalised aggressive periodontitis

Generalised aggressive periodontitis (GAP), formerly named generalised early-onset periodontitis, usually affects patients aged
under 30. GAP is characterised by the presence of generalised interproximal attachment loss affecting at least three permanent teeth
other than the first molars and incisors [11] (Figures 13 and 14).

Figure 13. Generalised aggressive periodontitis. The scarce amount of microbial deposits is conflicting with

the severity of periodontal tissue destruction as shown in Figure 14.

Figure 14. Full-mouth series periapical X-rays. Advanced bone destruction is evident.

The genetic bases of periodontitis
The observation that inheritance was an important component in the development of periodontal diseases was proposed as early as
1935 [12]. Investigations focused on genetic risk factors are currently characterising periodontal research in the genetic field.
Mutations and polymorphisms
DNA sequence variations are described as mutations and as polymorphisms. A mutation is defined as a change in a DNA sequence
away from normal. In periodontitis, specific mutations have been identified to cause the genetic basis of various syndromic
conditions (Table 1) [13]. Genetic mutations are deterministic of simple genetic diseases. However, these genetic diseases are rare
and do not characterise the most common forms of periodontitis. In fact, from a genetical point of view, probably the most common
forms of periodontitis arise as a result of single-nucleotide polymorphisms (SNPs). The arbitrary cut-off point between a mutation
and a polymorphism is 1%. That is, to be classed as a polymorphism, the least common allele must have a frequency of 1% or more
in the population. When the frequency is lower than this, the allele is regarded as a mutation. Numerous studies have investigated
SNPs in both the chronic and aggressive forms of periodontitis. The distribution of SNPs varied between ethnic groups. Ethnic
difference(s) in genes encoding the interleukins IL-1 and IL-6, Fc receptors (Fc RIIa, Fc RIIIa and Fc RIIIb), tumour necrosis factor
alpha (TNF-α), vitamin D receptor, CD14 and matrix metalloproteinase-1 (MMP-1) were found. Several studies investigated the
genes affecting the expression of interleukin-1, interleukin-6, tumour necrosis factor, interleukin-10, E-selectins, Fc-gamma
receptor, CD14, toll-like receptors and periodontal disease. The largest part of the studies shows no correlations between the
presence of disease markers and the tested SNP in both the aggressive and chronic forms of periodontitis [14]. It is important to
clarify that in complex disease, a given polymorphism is necessary but not sufficient to cause disease. In fact, the interplay of
genetic and environmental factors is fundamental in determining the disease phenotype. Hence, a single functional genetic
polymorphism is not sufficient to cause disease; consequently, such functional polymorphisms may be found in individuals with no
evidence of disease.

Table 1. Specific mutations cause the genetic basis of various syndromic conditions in which periodontitis is present
Bacterial burden: a challenge for periodontal tissues
The average 200-lb (90 kg) human body carries around with it about 6 lb (2.7 kg) of bacteria. Some of them live in the oral cavity
forming a huge source of bacteria: to give an idea, in 1 mm3(1 mg) of dental plaque, 108 bacteria are present. The Human Oral
Microbiome Database lists 1,200 predominant oral species, with the order of 19,000 phylotypes [15].
Dental plaque is a biofilm containing over 700 individual taxa of aggregated microorganisms embedded within a self-produced
matrix of extracellular substance composed of bacterial polymers and salivary and gingival exudate products (Table 2). The
heterogeneity of plaque gradually increases and includes large numbers of Gram-negative anaerobic species in gingivitis
(approximately 25%) and periodontitis (approximately 75%) as compared to healthy gingiva (approximately 13%) (Table 3).

Table 2. Principal constituents of dental plaque

Table 3. The most frequent microbial species isolated in healthy gingiva, gingivitis and periodontitis
Further and more recent studies have demonstrated that there are specific associations among bacterial species within dental plaque.

Five closely associated clusters have been reported (Figure 15):

1. The green cluster (Campylobacter concisus, Eikenella corrodens, Actinobacillus actinomycetemcomitans serotype a).
2. The yellow cluster made up of a group of streptococci (Streptococcus mitis, Streptococcus sanguis, Streptococcus oralis).
3. The purple cluster (Actinomyces odontolyticus, Veillonella parvula).
4. The red cluster (P. gingivalis, Tanerella forsythia, Treponema denticola).
5. The orange cluster (Fusobacterium nucleatum subspecies, Prevotella intermedia, Prevotella nigrescens, Peptostreptococcus
micros, Campylobacter rectus, Campylobacter showae,Campylobacter gracilis, Eubacterium nodatum, Streptococcus
constellatus, Fusobacterium periodonticum).

Figure 15. Specific associations among bacterial species within dental plaque characterise five closely

associated clusters.(Adapted from [16]).

Finally, Actinotmyces naeslundii genospecies 2 (Actinomyces viscosus), Selenomonas noxia and A. actinomycetemcomitans serotype
b did not cluster with other species [16].
Periodontopathogens also colonise non-dental surfaces such as the tongue dorsum, oral mucosa and tonsils, and for this reason,
periodontitis therapeutic measures have to eliminate periodontal pathogens in the whole mouth (Figure 16) [17].

Figure 16. Tongue dorsum brushing with 0.5%chlorhexidine gel.The red colour of the tongue is due to the

use of erythrosine pads that have the capability to reveal the presence of bacterial plaque on the teeth and soft tissue. Chlorhexidine

(0.12%) puffs on the tonsils and chlorhexidine (0.12%) mouth rinse are further procedures necessary to eradicate periodontal

pathogens from the whole mouth.

An epidemiologic study found out that close members of the same family were infected via saliva with A.
actinomycetemcomitans strains of the same biotype and serotype [18]. For this reason, prevention measures against periodontal
pathogens must include the entire family members in order to prevent cross-infection [19].
Even if there are no sufficient microbiological evidences that could help us distinguish the different forms of periodontitis, it is clear
that:

1. The chronic and aggressive forms of periodontitis are not monoinfections.

2. Some microbiota are more important than others as aetiological agents of periodontitis.

Periodontal tissues as a ‘battlefield’ in the struggle against oral bacteria

The host-microbial balance constitutes the situation in clinically healthy periodontal tissue. Plaque accumulation and immunitary
response can create an imbalance of the host-parasite relationship occurring in destructive periodontal lesion. In fact, the fight
among bacteria and immunocompetent cells can devastate the battlefield, that is, the periodontium.

For simplification, we arbitrarily divided the immunitary response in periodontal tissue into three different compartments
(epithelium, connective, bone) in which three main cells (polymorphonuclear neutrophils (PMNs), macrophages, osteoclasts) are
representative of three different topical moments (Figure 17).

Figure 17. The mortal fight among bacteria and immunocompetent cells. It can devastate the ‘periodontal

battlefield’ since defensive immunitary reaction could paradoxically contribute to the tissue destruction. Activated
polymorphonuclear leukocytes, indeed, can cause tissue damage as a result of a variety of enzymes and oxygen metabolites that are

released from their granules during the battle against microbes. Bacterial LPSs activate macrophages, lymphocytes and fibroblasts

which secrete lymphokines activating MMPs. Metalloproteinases are enzymes that degrade the connective extracellular matrix and

can be detected in gingival crevicular fluid. Finally, many substances (PGE2, IL-1, IL-6, TNF-α) secreted by Mø, fibroblasts,

plasma cells and T lymphocytes are primarily involved in osteoclastic activation via the RANKL-OPG expression system.
Epithelial compartment: PMN activation
PMN leukocytes represent the first line of defence forming a protective wall against microorganisms. Activated polymorphonuclear
leukocytes can cause tissue damage as a result of their accumulation in epithelial tissues. Further damages can be caused by a
variety of enzymes and oxygen metabolites that are released from their granules during the battle against microbes[20,21]. Oxygen
metabolites such as hydrogen peroxide (H2O2) and reactive oxygen radicals (OH−) that are released into the phagosome defensive
immunitary reaction could paradoxically contribute to the tissue destruction. As a consequence, the junctional epithelium becomes
filled with ulcers and allows the passage of bacteria and their products in the underneath connective tissue.
Connective compartment: macrophage activation
In the subsequent line of defence, macrophages (Mø) play a decisive role to restrict bacterial spreading in the connective tissue.
Macrophages are an important source of lysosomal enzymes, cytokines and inflammatory mediators such as IL-1, TNF-α, PGE2
and transforming growth factor beta (TGF-β).

IL-1 is the principal inflammatory mediator released by LPS-activated macrophages, lymphocytes and fibroblasts. IL-1 stimulates
Mø and fibroblasts to secrete PGE2; moreover, it causes osteoclastic differentiation and activation [22].
TNF-α, principally secreted by LPS-stimulated macrophages and lymphocytes, causes osteoclastic differentiation and
activation [23].
PGE2 causes vasodilatation, vasopermeability and resorption of the alveolar bone. IL-1, TNF-α and PGE2 stimulate fibroblasts and
Mø to release MMPs, urokinase plasminogen activator (u-PA), tissue inhibitor of metalloproteinases, PGE2, TGF-β and interleukin-
1 receptor antagonist. As described below, disease severity appears linked to the existent equilibrium among different involved
molecules. The u-PA causes plasminogen transformation in plasmin which activates MMPs, enzymes degrading the connective
extracellular matrix. They can be detected in gingival crevicular fluid, particularly during the activity phase [24].
Bone compartment: osteoclast activation
Inflammation progresses in the apical direction involving the bone tissue. It is important here to highlight that bacterial plaque never
gets in direct contact with the bone tissue and that ‘running away’ from the bacterial aggregate is always at least 2 mm in distance
from it. Many substances (PGE2, IL-1, IL-6, TNF-α) secreted by Mø, fibroblasts, plasma cells and T lymphocytes are primarily
involved in osteoclastic activation.

The receptor activator of NF-kB ligand (RANKL) is a recently described member of the tumour necrosis factor superfamily
promoting osteoclastic differentiation from haemopoietic precursors and the inhibition of osteoclast apoptosis. Under physiological
condition, RANKL produced by osteoblasts binds to RANK on the surface of osteoclast precursors. RANKL is up-regulated by
osteotropic factors such as PTH and IL-11. Osteoprotegerin (OPG), a member of the TNF receptor superfamily, is produced by
fibroblasts constituting a false target for RANKL [25,26]. Hence, OPG is an inhibitor of bone resorption. The balanced regulation of
the RANKL-OPG expression system can determine health from disease (Figure 18).

Figure 18. The RANKL-OPG expression system. Under physiological condition, RANKL produced by

osteoblasts binds to RANK on the surface of osteoclast precursors. OPG is produced by fibroblasts constituting a false target for

RANKL. The balanced regulation of the RANKL-OPG expression system can determine health from disease.
Environmental risk factors
Smoking and diabetes mellitus are the most frequent co-factors strongly associated with the aggravation of periodontitis. Other
situations such as obesity, stress and osteoporosis have been identified as co-factors in the progression of periodontitis [27].
Diabetes mellitus
The present percentage of diabetics is very high worldwide, and these numbers are increasing dramatically. It is not exaggerate to
claim that we are going to face a dramatic diabetic emergency in the next years (Figure 19).
 Figure 19. More than 371 million people have diabetes worldwide and the number is increasing in every

country. It is important to highlight that half of the people with diabetes do not know that they have it, and for this reason, the

majority of people who die from diabetes are under the age of 60. Nearly 5 million people died and US$471 billion were spent due

to diabetes in 2012. International organisations are going to face a dramatic diabetic emergency in the next years. (Adapted

from [28]).
The contemporary consensus is that diabetic patients are at increased risk of periodontitis [29]. Patients with type 1 (insulin-
dependent) and type 2 (non-insulin-dependent) diabetes mellitus have been found to be equally at risk for periodontitis [30]. The
severity of periodontitis has been proved to increase with the onset of diabetes at a younger age as well as with poorer metabolic
control of diabetes [31]. It has been claimed that periodontitis is the sixth complication of diabetes, together with retinopathy,
nephropathy, neuropathy, macrovascular diseases and altered wound healing [32]. Diabetes mellitus is the only systemic disease
positively associated with attachment loss with an odds ratio of 2.32 (95% confidence interval (CI) 1.17–4.60) [33]. Some authors
presumed a two-way relationship in which periodontal therapy can improve metabolic control in diabetic patients [34]. In these
studies, periodontal treatment was associated with a reduction in HbA1c levels, and moreover, inflammatory biomarkers decline
with periodontal treatment [35-37]. In contrast, non-significant reduction in HbA1c values was recorded in several studies [38-40].
Very recently, a meta-analysis of nine intervention studies of 485 people with diabetes concluded that periodontal treatment could
lead to a significant 0.79% (95% CI 0.19–1.40) reduction in HbA1c levels [41]. A recent Cochrane review on the treatment of
periodontal disease for glycaemic control in people with diabetes declared that further controlled studies are necessary to clarify the
topic [42]. These conflicting data are difficult to understand in order to clarify the influence of periodontitis in glycaemic control.
Hence, supplementary controlled clinical trials appear urgent and necessary to definitely assess if periodontal therapy can improve
metabolic control in diabetic patients.
Smoking
Over the past decades, a multitude of papers about the relationship between smoking and periodontitis have been published. The
contemporary consensus is that:

1. Cigarette smoking is associated with a relative risk, ranging from 2.05 (95% CI 1.47–2.87) for light smokers increasing to 4.75
(95% CI 3.28–6.91) for heavy smokers, of developing periodontitis [33,43].
2. The negative effect of smoking is dose dependent and cumulative [44].
3. The negative effect of smoking is marked in younger individuals [45].
4. Smoking affects the healing potential of periodontal tissues [46].
5. Smoking is associated with the recurrence of periodontitis during periodontal maintenance[47].
Obesity, stress and osteoporosis
Other conditions such as obesity, stress and osteoporosis have been involved as co-factors in the progression of periodontitis, even if
the association appears weak and still debatable.

Obesity
It has been suggested that obesity is a strong risk factor for periodontal tissue destruction [48] since adipose tissue represents much
more than a fat accumulation. It produces cytokines and hormones, collectively called adipokines or adipocytokines, which may
play a key role in modulating periodontitis [49].
An association between obesity and periodontal disease in humans was reported for the first time by Saito et al. [50]. The authors
estimated that the relative risk for periodontitis was 3.4 in persons with a body mass index of 25–29.9 kg/m2 and 8.6 in those with a
body mass index of >30 kg/m2. These results were confirmed by other authors [51,52]. Genco et al. [53] demonstrated that the
severity of periodontal attachment loss was modulated by insulin resistance. In addition, it was reported that maintaining a normal
weight was associated with a poorer frequency of periodontitis [54,55].
Stress
The impact of stress on periodontal diseases has not yet been clarified. Stressful life events have been shown to modulate the
endocrine and immune systems. Stressful life events could affect periodontal disease progression through (1) unhealthy behaviours
(poor oral hygiene, increased tobacco smoking) and (2) pathophysiological factors (higher glucocorticoid and catecholamine levels)
which affect bacterial, immunological, inflammatory and hormonal profiles, leading to an increased susceptibility to periodontal
disease [56,57]. Finally, in a systematic review, a positive relationship between stress and chronic periodontitis was confirmed [58].
Osteoporosis
Osteoporosis is a metabolic bone disorder characterised by the loss of bone mineral density, principally recorded in postmenopausal
women. It has been proposed that osteoporosis could affect the alveolar bone leading to rapid resorption in periodontal women. In
one study, 189 postmenopausal women were controlled over a 7-year period. An association between the loss of bone mineral
density and increased risk of additional tooth loss was reported. In a review, it has been shown that 7 out of 17 studies reported a
positive relationship between osteoporosis and clinical attachment loss. Eleven out of 19 studies found a positive association
between osteoporosis and tooth loss [59]. Other studies showed negative or equivocal results [60].
It can therefore be concluded that since many of the studies were uncontrolled and had small sample sizes, the validity of their
conclusions needs to be confirmed. Thus, the association between osteoporosis and periodontitis in humans remains weak and still
debatable [61].
Periodontology approaches the future: 5Ps for five diagnostic levels
In addition to the traditional instruments for periodontal diagnosis, in the next future, well-organised population screening protocols
utilising chairside diagnostic biomarkers for periodontal disease will be disposable. With reference to this, the last section of the
present paper will be focusing on the diagnostic tools currently utilised for periodontal diagnosis (the present time) and on the most
promising diagnostic tools (i.e. biomarkers and high-tech instrumentations) that are going to enter in clinical periodontology (the
next future).
The present time: a precise picture of a single periodontal patient's existing condition
Diagnostic imaging and periodontal charting provide a complete description of the patient's periodontal condition.

Diagnostic imaging: a fundamental step to assess the periodontal conditions of a single patient
Full-mouth high-definition digital photographs By the use of a high-resolution professional digital camera, the operator takes a
series of five pictures (frontal, right lateral, left lateral, palatal and lingual sides) during the initial visit. Since the camera is used in
tandem with a computer screen, we can, in real time, easily show the patient the recorded images to illustrate his/her dental and
periodontal health. The camera is not only a diagnostic tool but also a fantastic educational aid in helping us to reinforce
the compliance of the patients, one of the most important topics in participatory periodontology. Finally, ‘before and after’ pictures
can give periodontists and patients an objective representation of periodontal health improvement (Figure 20).

Figure 20. Full-mouth high-definition digital photographs. By the use of a high-resolution professional

digital camera, the operator takes a series of pictures during the initial visit. Before and after pictures can give periodontists and

patients an objective representation of periodontal health improvement. Thus, the camera is a fantastic educational aid to reinforce

the compliance of the patients and a diagnostic tool for the periodontist.
Full-mouth series periapical X-rays
An intra-oral X-ray provides a clear picture of the state of the patient's individual tooth from the crown to the tip of its root
(Figure 21). Moreover, it provides information on the height and configuration of the interproximal alveolar bone. A full-mouth X-
ray series is an important diagnostic support in periodontal patients (14/16 periapical X-rays). Full-mouth series periapical X-rays
create a full view of the patient's teeth and surrounding bone tissue which must be combined with a meticulous assessment of
periodontal charting in order to make a correct evaluation regarding ‘horizontal’ and ‘angular’ bony defects.

Figure 21. A full-mouth X-ray series. It is an important diagnostic support in periodontal patients (14/16

periapical X-rays) since it creates a full view of the patient's teeth and surrounding bone tissue.
Periodontal charting: a complete status of the patient's periodontal health
Periodontal charting (full-mouth plaque score, full-mouth bleeding score, probing depth, clinical attachment level, bleeding on
probing, recessions, mobility, migration, halitosis) provides a complete picture of periodontal conditions of a single patient
(Figure 22) [3]. Measurements are accomplished with a calibrated periodontal probe (Figure 23) inserted into the sulcus and in a
parallel position with respect to the long axis of the tooth (Figure 24).
 Figure 22. Periodontal charting provides a complete picture of the periodontal conditions of a single

patient. (Adapted from [62]).

Figure 23. Calibrated periodontal probes are routinely used for periodontal screening.

Figure 24. A periodontal probe. It is inserted into the sulcus and in a parallel position with respect to the

long axis of the tooth. The physiological value of PPD is considered to be ≤3 mm. PPD allows an immediate evaluation of diseased

sites.
Full-mouth plaque score
The full-mouth plaque score is defined as the percentage of sites where plaque is present divided by the number of sites examined.

Full-mouth bleeding score

The full-mouth bleeding score is defined as the percentage of sites bleeding with respect to the number of sites examined.

Probing pocket depth

Probing pocket depth (PPD) is the distance from the gingival margin to the bottom of the gingival sulcus/pocket. It is measured by
means of a graduated periodontal probe with a standardised tip diameter of 0.5 mm. Measurement is taken for each tooth at the
mesio-buccal line angle, the mid-buccal, the distobuccal line angle, the distolingual line angle, the mid-lingual and the mesio-lingual
line (six sites for each tooth) (Figures 25 and 26). The physiological value of PPD is considered to be ≤3 mm. PPD allows an
immediate evaluation of diseased sites.

Figure 25. PPD and CAL measurements. They are taken for each tooth at (left to right) the mesio-buccal

line angle, the mid-buccal, the distobuccal line angle, the distolingual line angle, the mid-lingual and the mesio-lingual line.

Figure 26. PPD, CAL and REC measurements. PPD (blue line) is the distance from the gingival margin to

the bottom of the gingival sulcus/pocket. CAL (green line) is assessed by means of a graduated probe and expressed as the distance

in millimetres from the CEJ to the bottom of the periodontal pocket. REC (orange line) is defined as the apical migration of the

gingival margin. It is measured from the cement-enamel junction (curved yellow green line) to the gingival margin.
Clinical attachment level
Clinical attachment level (CAL), formerly called probing attachment level, is assessed by means of a graduated probe and expressed
as the distance in millimetres from the cement-enamel junction (CEJ) to the bottom of the periodontal pocket (Figures 25 and 26).
The severity of the attachment loss may be considered mild (CAL = 1–2 mm), moderate (CAL = 3–4 mm) or severe (CAL ≥ 5 mm).
Recessions
Recession (REC) is defined as the apical migration of the gingival margin. In most cases, it is due to gingival inflammation or
incorrect (traumatic) tooth brushing. It is measured from the cement-enamel junction to the gingival margin by the use of a
periodontal probe (Figure 26).
Bleeding on probing
A periodontal probe is inserted at the ‘bottom’ of the gingival sulcus or periodontal pocket. Blood coming out from the bottom of
the pocket can be recorded during probing (Figure 27). Bleeding on probing (BoP) is currently the unique predictive test routinely
used for monitoring disease progression or periodontal stability (discussed in the next sections).

Figure 27. Blood coming out from the bottom of the pocket can be recorded during probing (BoP+).
Mobility and migration
Unphysiological mobility and migration are generally due to the reduction of periodontal support caused by bone resorption in
consequence of periodontitis. Physiological forces (tongue, lips, occlusion, etc.) can cause the movement and migration of the tooth
with reduced periodontium.

Halitosis
Halitosis is defined as the presence of unpleasant breath odour. Gram-negative bacteria are the primary pathogens responsible for
oral malodour production. Other causes of halitosis are uncontrolled diabetes, gastrointestinal diseases, renal failure and diseases
affecting the upper/lower respiratory tract.
Unfortunately, periodontists can get only few predictive information about the progression and none about the rise of the disease
from the tools described above. BoP is currently the unique predictive test used by periodontists for routinely monitoring disease
progression or periodontal stability. BoP repeatedly positive (BoP+) is a predictor of future loss of attachment (activity phase) in
30% of cases (positive predictive value), meanwhile BoP repeatedly negative (BoP−) is a predictor of periodontal health in 98% of
cases (negative predictive value) [63-65]. In addition to that, a functional diagram to evaluate the patient's risk for recurrence of
periodontitis (‘spider's web’) has been proposed (Figure 28). It consists of an assessment of the level of infection (full-mouth
bleeding score), the prevalence of residual periodontal pockets, tooth loss, an estimation of the loss of periodontal support in
relation to the patient's age, an evaluation of the systemic conditions of the patient and finally an evaluation of environmental and
behavioural factors such as smoking. All these factors should be contemplated and evaluated together [66]. Bearing in mind what
has been discussed above, it appears clear that, at present, a periodontal defence strategy is almost totally reactive: periodontists take
action generally when periodontitis has already begun in periodontium destruction. In order to face mild or advanced periodontal
lesions, periodontists are currently able to put in place sophisticated periodontal therapeutic strategies, but this does not seem
enough. Recently, researches are gradually giving us the instruments to switch the therapeutic point of view from the current
reactive to a more advanced predictive model (Figure 29).

Figure 28. Spider's web. It consists of an assessment of the level of infection of a single patient contemplated

and evaluated together. In the present case, a heavy-smoker 50-year-old patient presents a high periodontal risk (30 BOP + sites, 32

sites with PPD ≥ 5 mm). (Adapted from [67]).

 Figure 29. The aim of predictive, preventive, personalised, participatory periodontology. The aim is to

transform the actual reactive therapeutic point of view, in which tissues destruction is clinically detectable, into a futuristic

predictive one in which the disease is early intercepted when it is already in a sub-clinical phase.
The next future: hi-tech diagnostic tools and specific biomarkers to detect early periodontal
damage
Knowledge in dentistry is estimated to double every 4–5 years in comparison with the 1950s when it was estimated to take 25 years
for such an expansion [68]. Enhancement in dental knowledge revealed genetic, microbiological and immunological mechanisms at
the base of periodontal diseases. Point-of-care (POC) testing allows rapid diagnostic tests in which results can be obtained
immediately rather than waiting days for outside lab results to arrive [69]. Chairside tests (CSTs) belong to POC cluster of analysis.
They can give an immediate indication on the dental health of a single patient to dental operators. CSTs based on saliva, gingival
crevicular fluid, cell and bacteria sampling are going to be routinely used by periodontists for a novel approach to the diagnosis,
monitoring, prognosis and management of periodontal patients. In the larger healthcare community, ‘dentists and oral health
professionals may be positioned to expand the reach and impact of preventive medicine through the application of cost-effective and
non-invasive oral fluid screening tests and referring patients for necessary medical care’ [70].
The first cause of tooth loss in industrialised world is periodontitis which is the result of the interaction between genetic tendency
and environment influence. In order to understand the growing value of the 5Ps, we have to consider some data that are currently
arising:
1. The European population is becoming progressively older.

2. Periodontitis generally strikes people older than 40 years.

3. Periodontitis can cause serious detriment of the stomatognathic organ.

It appears clear, therefore, that periodontitis has to be considered as a social disease since it affects millions of people in Europe, and
consequently, strategies have to be organised by national and international health organisations in order to intercept and treat the
disease before it can create serious damages to a large part of the European population. A similar situation has been recorded in the
USA in which 31% of the population exhibited mild forms of periodontitis, 13% displayed periodontitis of moderate severity and
4% suffered from advanced periodontitis [71]. In order to face this situation, we should modify our approach towards diseases.
Today, the work of periodontists is considered as ‘a reactive effort’ in the sense that we wait until the patient is sick before
responding; on the contrary, the futuristic 5Ps focuses on the early integrated diagnosis (genetic, microbiology, host-derived
biomarker detection) with the intention to detect periodontitis at an earlier stage, when it is easier to be treated successfully.
Here, we intend to propose five diagnostic levels (high-tech diagnostic tools, genetic susceptibility, bacterial infection, host
response factors and tissue breakdown-derived products) to be evaluated with the intention to obtain a clear picture of the
vulnerability of a single individual to periodontitis in order to organise patient stratification in different categories of risk
(Figure 30).

Figure 30. 5Ps flow chart. Five levels characterise a futuristic approach for periodontal diagnosis. The first

level is represented by high-tech diagnostic tools such as LOC and CBCT. In the next future, LOC will be able to give us genetic,

microbiological and host-derived information in real time. Co-factors (e.g. diabetes, osteoporosis) will be detected by the use of

dedicated high-tech chairside diagnostic tools. Moreover, a detailed bone tissue morphology is revealed by low-radiation digital

computed tomography which offers a digital volume composed of three-dimensional voxels that can then be manipulated with

specialised software. The second level will provide useful information about the genetic susceptibility of a single patient, while the

third level will give us the presence of causative bacterial factors in dental plaque. Finally, host-derived biomarkers (host response
factors and factors derived from periodontal tissue breakdown) will be chairside-detected in order to early intercept periodontal

destruction.
First diagnostic level: (lab-on-a-chip, gas chromatographs, cone beam computed tomography)
High-tech diagnostic tools will give periodontists the possibility:

1. To identify a periodontal initial lesion when it is not yet clinically detectable.

2. To intercept the so called ‘active phase’ of periodontitis.

Lab-on-a-chip prototypes, gas chromatographs and cone beam computed tomography are three categories of high-tech devices that
will be used everyday for the diagnosis of periodontitis in the not too distant future.

Lab-on-a-chip
A lab-on-a-chip (LOC) is a device that integrates several laboratory functions on a single chip of only millimetres in size
(Figures 31 and 32).

Figure 31. Lab-on-a-chip micronised pulse oximeter. Until a few years ago, the diagnostic tool shown in

the picture was sensibly bigger than the current one, and for this reason, it could be used only in hospitals. Nowadays, thanks to the

reduced dimensions, the oximeter can be lent from hospitals to patients, who can so check daily their oxygen absorption in their

own houses.

Figure 32. Probably the first chairside lab-on-a-chip utilised was the illustrated tool to check glycaemic

level. This instrument can be useful for initial diabetes screening in patients at risk. By the use of the patient's single blood drop, the

operator can inspect, in a few minutes, the actual glycaemic level in the patient's blood.
LOCs deal with the handling of extremely small fluid volumes down to less than picolitres (microfluidics). Microfluidics represent
the technology behind a new miniaturised analysis system for biological applications such as DNA amplification, purification and
separation [72]; sequencing[73]; proteomic analysis [74]; and single-cell gene expression profiling [75]. The use of microfluidic
devices has a number of significant advantages such as smaller sample requirement (usually several nanolitres), reagents come with
the chip and reduced reagent consumption (especially significant for expensive reagents, which is an important concern in clinical
laboratories today) that means an immediate indication on the periodontal health of a single patient to dental operators [76]. Finally,
the fabrication techniques used to construct microfluidic devices are relatively inexpensive and very open to mass production.
Gas chromatographs
Halitosis is a major concern to the general public and the source of a multi-million-dollar industry worldwide [77]. Many patients
affected by oral malodour often remain completely unaware of this fact, while others complain of halitosis even if no objective basis
can be found: this situation has been defined as the ‘bad breath paradox’. Halitosis is caused by physiologic or pathologic
conditions. Physiologic halitosis (the so-called ‘morning breath’) is caused by the stagnation of saliva that disappears with drinking,
consumption of food or tooth brushing.
Pathologic halitosis is principally caused by volatile sulphur compounds (VSCs), a family of catabolites resulting from oral bacterial
activity. The most important determinants of malodour are hydrogen sulphide (H 2S) and methyl mercaptan (CH3SH), which are
catabolites of cysteine and methionine. Other volatile components are aromatic compounds resulting from the degradation of
tryptophan (indole and skatole), short-chain fatty acids (acetic and propionic) and some polyamines (cadaverine and putrescine)
(Table 4) [78]. The production of volatile sulphureous compounds is mainly derived by the putrefaction of food debris, cells, saliva
and blood within the oral cavity mainly through microbial activity [79]. The intensity of clinical bad breath is significantly
associated with the amount of intra-oral VSCs [80]. Gram-negative bacteria are the primary pathogens responsible for oral
malodour production [81]. Patients with periodontal diseases often complain of oral malodour since the periodontal pocket is an
ideal environment for VSC production with respect to the bacterial profile and sulphur source. Other authors demonstrated that a
higher amount of VSCs was highly correlated with probing pocket depth, clinical attachment level, bleeding on probing,
radiographic bone loss and Gram-negative pathogen species (T. denticola, P. gingivalis, P. intermedia) [82]. The most common
device used to evaluate halitosis is the Halimeter® (Interscan Corp., Chatsworth, CA, USA) that measures volatile sulphur
compounds in exhaled air. The Halimeter® does not measure other important odorants, such as volatile fatty acids and cadaverine,
which are involved in oral halitosis: this could lead to a false negative result when malodour can be detected by the examiner, but
the volatile sulphur compound levels are in the low range [83]. A portable gas chromatograph named Oral Chroma™ (Abilit Corp.,
Osaka, Japan) has been introduced to detect VSCs [84].

Table 4. Principal volatile components responsible for oral pathologic halitosis

The Halimeter® has been shown to be more sensitive to H2S than to methyl mercaptan and almost insensitive to dimethyl sulphide,
whereas the Oral Chroma™ measures all three gases with equally high sensitivities [85].
Cone beam computed tomography
Multi-slice computed tomography (MSCT) is a medical imaging technique using a narrow fan beam that rotates around the patient's
head acquiring thin axial slices. During these repeated rotations, MSCT emits a high radiation dose, and it leaves a gap of
information between each rotation. Consequently, the software must connect together the images and calculate what is missing.
Cone beam computed tomography (CBCT) technology was first introduced in the European market in 1996 and into the US market
in 2001 [86]. CBCT uses a cone-shaped beam (the X-rays are divergent) to acquire the entire image in a scan using only one
rotation. During a CBCT scan, the scanner rotates around the patient, obtaining up to almost 600 separate images. The scanning
software collects the anatomical data and produces a digital volume composed of three-dimensional voxels (instead of traditional
pixels) that can then be visualised and manipulated with specialised software. The result is a more precise image without missing
information (Figure 33).

Figure 33. MSCT. It collects the anatomical data and produces a digital volume composed of three-

dimensional voxels that can then be visualised and manipulated with specialised software. A three-dimensional reconstruction of the

upper and lower maxillae can be obtained, and anatomical structures can be easily inspected.
Second diagnostic level: genetic susceptibility
The largest part of the studies shows no correlations between the presence of disease markers and the tested SNPs in both the
aggressive and chronic forms of periodontitis [14]. The polymorphisms that seemed to be linked with periodontitis in different
ethnic groups were associated with the Fc-gamma receptor genes. However, these polymorphisms of the same gene were found in
both chronic periodontitis and aggressive periodontitis [87,88]. A weak association between the SNP in interleukin-1 genes and
chronic periodontitis was found in a recent meta-analysis [89]. Interleukin-1 is a pro-inflammatory agent that is released by
macrophages, lymphocytes, platelets and endothelial cells. The gene encoding this cytokine is assigned to chromosome 2q13–
21 [90].
In 1997, Kornman et al. described a composite genotype formed by two polymorphic loci - interleukin-1A (−889) and interleukin-
1B (+3954) - which are single-nucleotide polymorphisms that carry a C-T transition [91]. Interleukin-1A (−889), however, was
outdated by the investigation of the interleukin-1A (+4845) G-T dimorphism, in which the two loci comprising the periodontitis-
associated genotype were found to be in linkage disequilibrium [92].
Therefore, the analysis of the interleukin-1A (+4845) single-nucleotide polymorphism provides the same genetic information [93].
Several studies have evaluated the utility of the commercially available IL-1 genetic susceptibility test (Figure 34) [94].
Unfortunately, we do not have any sufficient size-controlled studies that would allow us to evaluate the efficacy of the IL-1
genotype[95,96]. Thus, although certain studies are encouraging, there are currently insufficient data to support a modification of
treatment protocols for chronic periodontitis patients based on IL-1 testing [97].

Figure 34. PST-positive genotype test interleukin-1A (+4845) and interleukin-1B (+3954). Several studies

encourage the routine application of such tests to assess periodontal risk in a single patient.
Third diagnostic level: bacterial infection
Haffajee and Socransky [98] suggested six types of lines of evidence to be used to support an aetiological role for bacteria in
periodontal infections:
1. Elevated odds ratio in disease.

2. Conversion of disease to health when bacteria are suppressed.

3. Development of a host response.

4. Presence of virulence factors (capability to avoid host defences and to damage tissues).

5. Evidence from animal studies corroborating the observations in humans.

6. Support from risk assessment studies.

Following the above criteria, the consensus report of the World Workshop on Periodontitis [99] identified three bacterial species for
which sufficient data have accumulated as causative factorsfor periodontitis: A. actinomycetemcomitans (recently renamed
to Aggregatibacter actinomycetemcomitans) [100], P. gingivalis and Bacteroides forsythus (renamed to Tanerella forsythia) [101].
The consensus report stated that A. actinomycetemcomitans is most often found in aggressive (‘early-onset’) periodontitis,
whereas P. gingivalis and T. forsythia are found more frequently in chronic (‘adult-onset’) periodontitis. Moderate evidence to
support an aetiological role was reported for C. rectus, E. nodatum, P. intermedia, P. nigrescens, Parvimonas
micra (formerlyMicromonas micros and Peptostreptococcus micros), the Streptococcus intermedius complex and T. denticola.
Finally, an initial evidence included on the list of probable periodontal pathogens E. corrodens, enteric
rods, Pseudomonas species, Selenomonas species and Staphylococcus species. This report received general acceptance by the
periodontal community and is still regarded as valid.
Even if there are no sufficient microbiological evidences that could help us in distinguishing the different forms of periodontitis, it is
clear that:

•The chronic and aggressive forms of periodontitis are not monoinfections.

•Some microbiota are more important than others as etiological agents of periodontitis.

For these reasons, it appears clear that the microbial testing of sub-gingival plaque could be a valid support for a correct diagnosis of
periodontitis. The anaerobic culture test is the most sophisticated technique to analyse the composition of sub-gingival plaque. All
cultivable microbial species in the sub-gingival sample can be detected, and proportions of the various pathogens can be established.
Anaerobic culture testing allows the antimicrobial susceptibility testing of periodontal pathogens. Anaerobic culture testing is
advised especially in the case of refractory periodontitis, atypical forms of pathogens or periodontitis, peri-implantitis and
immunocompromised patients. In routine cases, a DNA-based chairside test (semi-quantitative polymerase chain reaction (PCR)) is
indicated. Bacteria do not need to be viable; consequently, time is not an issue with the present test. The number of target bacteria is
determined semi-quantitatively (0 to +++).

CSTs for bacteria detection provide information about the presence and relative importance of putative pathogens.

The periodontist has to follow the following steps in order to perform a correct DNA-based chairside test (semi-quantitative PCR)
for bacterial plaque analysis:

•Meticulous removal of supra-gingival plaque.

•Sampling of sub-gingival plaque by the insertion of sterile paper points into the deepest pockets in each quadrant.

•Sending samples to a specialised laboratory.

The specialised laboratory will perform the DNA examination and identification of bacterial species. The number of target bacteria
is determined semi-quantitatively (0 to +++) (Figure 35). Since optimal plaque control by the patient is of paramount importance for
a favourable clinical and microbiologic response to therapy, microbiological analysis laboratory results should be discussed with
patients in order to reinforce their compliance. Patients have to be placed on an individually tailored maintenance care programme,
including the instruction of oral hygiene, in order to obtain optimal plaque control and continuous evaluation of the occurrence of
disease progression.

Figure 35. Consecutive steps to perform a correct DNA-based chairside test (semi-quantitative PCR)

for bacterial plaque analysis. Clinical diagnosis (chronic periodontitis or aggressive periodontitis), cigarette smoking, systemic

pathologies and antibiotic consumption are the initial information requested. Then, a meticulous removal of supra-gingival plaque

has to be performed. After that (see the figure clockwise), periodontal charting detects four different sites showing the deepest

probing pocket depth whose values were reported on a DNA-PCR form. Clinical attachment level measures of the selected sites are

also requested. In the present case, pyorrhea was present (blue arrow). Samples of sub-gingival plaque are carried out by the

insertion of sterile paper points into the deepest pockets in each quadrant. Paper points are then stored into a vial and the samples

sent to a specialised laboratory to perform the DNA examination and identification of bacterial species, together with the DNA-PCR

form. The number of target bacteria is determined semi-quantitatively (0 to +++) and sent to the periodontist together with the

diagnosis and therapy advice. Results are useful for the periodontist who will have a picture of a single patient's microbiological

infection and for the patients in order to reinforce their compliance. Finally, the periodontist, having considered the species found,

should propose an individually tailored maintenance care programme to a single patient.

Fourth and fifth diagnostic levels: host response factors and tissue breakdown-derived products
At present, well-studied molecules associated with host response factors and with derived tissue destruction mediators have been
proposed as diagnostic biomarkers for periodontitis [102]. Many dental associations, such as the American Dental Association
(ADA), recognise the importance of continued research on oral fluid diagnostics and welcome the development of rapid point-of-
care tests that provide accurate measurements of clinically validated biomarkers. The ADA council ‘encourages dentists to take
leadership roles in integrating the tests and related technologies into clinical practice, consistent with the best available scientific
evidence’ [70].
We are going to discuss inflammatory/immunological reaction and sub-clinical tissue destruction in the same section since they:

1. Happen approximately at the same time.

2. Share the same modality of non-invasive sample collection.

3. Release biomarkers which can be detected in the same diagnostic medium (oral fluid, gingival crevicular fluid)

Oral fluid (whole saliva) as a diagnostic tool

Probably the simplest organic diagnostic tool is oral fluid, a watery substance with multiple functions. Oral fluid or whole saliva is
composed of 99.5% water, while the other 0.5% consists of antibacterial compounds. The advantages of using oral fluid as a
diagnostic medium for a rapid point-of-care testing include non-invasive sample collection, simplicity of access and acceptance by
patients. Oral fluid (also called as whole saliva) is the fluid obtained from the mouth by expectoration. It includes glandular-duct
saliva and gingival crevicular fluid:
1. Glandular-duct saliva: saliva secreted by the parotid, sub-mandibular, sub-lingual and minor salivary glands (2,000 ml/24 h) is
obtained directly from the glandular ducts with specially designed collectors. Glandular-duct saliva contains predominantly
secretory IgA.

2. Gingival crevicular fluid (GCF) is an exudate flushing from the gingival sulcus (0.5 to 2.5 ml/24 h). GCF is a versatile and non-
invasive means to sample the biomarkers of inflammation and bone resorption in the oral cavity. GCF represents serum components
overlaid with products from local physiologic or pathologic phenomena. In particular, pathologic phenomena such as connective
tissue destruction and bone loss may have a diagnostic value[103,104]. Whilst gingival crevicular fluid is the most appropriate
diagnostic medium to use in analyses, it appears clear that the use of whole saliva is more practical even if reactants need to be
highly sensitive since biomarkers are more diluted [105,106].
Salivary biomarkers for periodontal disease
Recently, the entire human salivary proteome was reported by a consortium of three research groups [107], and this revealed that
1,166 proteins are present in human saliva [108]. Over 65 GCF components have been examined as possible markers for the
progression of periodontitis (for a complete review, see [109]). These components fall into three general categories: (1) host-derived
enzymes and their inhibitors, (2) inflammatory mediators and host response modifiers and (3) tissue breakdown products. We have
searched the literature for more promising components of gingival crevicular fluid in regard to potential diagnostic value for
periodontitis (Table 5) (for a complete review, see [110]).

Table 5. Most promising salivary biomarkers for the diagnosis of periodontal disease
Alkaline phosphatase (host-derived enzyme)
Alkaline phosphatase is an enzyme produced principally by neutrophils and then by fibroblasts, osteoblasts, osteoclasts and several
bacteria. It plays a role in the physiological turnover of the periodontal ligament, root cement and alveolar bone. The amount of
alkaline phosphatase in gingival crevicular fluid samples appears higher in the active sites than in the inactive sites. Moreover,
elevated alkaline phosphatase levels preceded attachment loss, while no clinical parameters were yet discriminatory [111].
Beta-glucuronidase (host-derived enzyme)
Beta-glucuronidase is a lysosomal enzyme that could be thought as an indicator of periodontal disease activity. Lamster et al. [112]
showed a predictive value for beta-glucuronidase in relation to clinical attachment loss. Nakashima et al. [111] reported that beta-
glucuronidase was significantly higher in active vs. inactive sites.
Cathepsin B (host-derived enzyme)
Cathepsin B is an enzyme active in proteolysis. Macrophages are the cellular source of cathepsin B in gingival crevicular
fluid [113]. Cathepsin B levels (1) have been found to be increased in periodontitis but not in gingivitis, (2) were higher in rapid loss
sites than in paired control sites and (3) appeared reduced after periodontal treatment [114-116].
MMP-8 (collagenase-2) (host-derived enzyme)
MMP-8 in gingival crevicular fluid has latent and active forms. The latent enzyme may be present in gingivitis and the active form
in periodontitis. MMP-8 appears 18-fold higher in progressing periodontitis vs. stable periodontitis [117]. Mancini et al. proposed
the use of MMP-8 levels in gingival crevicular fluid as a test for active periodontal destruction [118].
MMP-9 (gelatinase) (host-derived enzyme)
MMP-9 appears elevated in subjects affected by advanced periodontitis associated with red complex anaerobic periodontal
pathogens (e.g. P. gingivalis and T. denticola) [119]. Samples from patients with recurrent attachment loss showed a twofold
increase of mean active MMP-9, and these levels decreased significantly following adjunctive metronidazole therapy [120].
Dipeptidyl peptidases II and IV (host-derived enzyme)
Neutrophils, lymphocytes, macrophages and fibroblasts are the main sources of dipeptidyl peptidases II and IV. Their main function
lies in the activation of the pro-forms of cytokines and enzymes and in the degradation of collagen tissue. Higher levels of both
enzymes in sites with rapid and gradual attachment loss were reported with respect to sites without attachment loss[121].
Elastase (host-derived enzyme)
Elastase is a proteinase released from the azurophilic granules of neutrophils and from macrophages (also called MMP-12). Elastase
has been recorded in GCF from periodontal patients at elevated levels and reduced after periodontal treatment. Many authors [122-
124] observed higher elastase levels in sites demonstrating progressive attachment loss in comparison with inactive sites.
RANKL/OPG/RANK system (host response modifiers)
The RANKL/OPG/RANK system can be detected in the gingival tissue, GCF and saliva. In the course of periodontitis, RANKL is
secreted by osteoblasts, fibroblasts, bone marrow stromal cells and activated T and B cells. Under physiological condition, RANKL
produced by osteoblasts binds to RANK on the surface of pre-osteoclasts. RANKL is up-regulated by osteotropic factors such as
OPG. RANKL is increased whereas OPG is decreased in periodontitis compared to healthy gingiva or gingivitis [125].
1-CTP (tissue breakdown products)
Pyridinoline cross-links represent a class of collagen-degrading molecules that include pyridinoline, deoxypyridinoline, N-
telopeptides and C-telopeptides. The role of pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (1-CTP) levels
in gingival crevicular fluid as a diagnostic marker of periodontal disease activity has been investigated by several studies. High
levels of 1-CTP were strongly correlated with clinical parameters and putative periodontal pathogens. Results showed that 1-CTP
appeared as a good predictor of future alveolar bone and attachment loss and demonstrated significant reductions after periodontal
therapy [126].
C-4-S (tissue breakdown products)
Chondroitin-4-sulphate (C-4-S) is the most common glycosaminoglycan in untreated chronic periodontitis sites, as shown in both
animal and human studies. Elevated glycosaminoglycan concentrations were also found in aggressive periodontal diseases, and
associations have been made with periodontal pathogens such as P. gingivalis[126]. A statistically significant correlation between
the GCF content of C-4-S, a bone-specific glycosaminoglycan, and PPD and CAL was reported [127].

Conclusions
Oral fluid is the mirror of periodontal health. It is a medium for clinically relevant information since it contains biomarkers specific
for periodontal diseases. Although the periodontal diagnostic value of oral fluid has been recognised for some time, most scientific
papers in the recent past have failed to support consistent aids to the clinician in periodontal diagnosis and therapy. Advances in
microfluidics technology are revolutionising molecular biology procedures for enzymatic analysis, DNA analysis and proteomics.
The evolution of microfluidics, digital microfluidics, appears promising for future application to diagnose periodontal diseases and
to prognosticate periodontal treatment.
Lab-on-a-chip technology may soon become an important part of efforts to improve worldwide periodontal health [128]. In
developed nations, the most highly valued qualities for portable, easy-to-use diagnostic tools include speed, sensitivity and
specificity; while in the underserved communities, resource-poor areas and poor countries, the goal of researchers is to create
microfluidic chips that will allow healthcare providers in poorly equipped hospitals [129] (Figures 36and 37).

Figure 36. A 24-year-old patient from Nigeria suffering from generalised aggressive

periodontitis. Periodontal diagnosis was effected in Naples (Italy) when the disease had already destroyed up to 80% of the

periodontal supporting bone.

Figure 37. A panoramic X-ray of the same patient. An advanced generalised destruction of the supporting

bone tissue is evident. One of the most important topics in periodontal diagnosis in the next future will be to create microfluidic

chips allowing healthcare providers in poorly equipped hospitals and areas of the world.
The use of LOC devices for periodontal inspection will involve less education than current diagnostic procedures and allow patients
to be screened for periodontal disease in settings other than the periodontist practice, such as at general practitioners, general
dentists or dental hygienists [130].
All these benefits make the lab-on-a-chip technology ideal for predictive, preventive, personalised and participatory periodontology.
The 5Ps represents with no doubt the future of our profession. Personalised therapy with tailored respect to the particular medical
reality of the specific stratified patient will be the ultimate target to be realized by the 5Ps approach. A long distance has to be
covered to reach the above targets, but the pathway has already been clearly outlined: it is ‘time for new guidelines in advanced
healthcare’ in dentistry too [131].

Consent
Written informed consent was obtained from the patients for the publication of this report and any accompanying images.

Competing interests
The authors declare that they have no competing interests.

Authors’ contributions
CC and SM conceived the present paper and participated in its draft. Both authors read and approved the final manuscript.

Authors’ information
CC is a researcher and professor at the University of Naples “FEDERICO II”. SM is a full professor and the Chairman of the
Degree Course in Dentistry at the University of Naples “FEDERICO II”.

Acknowledgements
The authors wish to thank the International Diabetes Federation for giving permission to reproduce Figure 19 and Prof. Dr.
Christoph A. Ramseier, Department of Periodontology, University of Berne, Switzerland for giving permission to reproduce
Figures 22 and 28. The authors thank Dr. Pierpaolo ‘Pirre’ Ballone for the drawings, the dental student Michele Colamaio who
helped in the digital organisation of the figures and Dr. Claudia Meoli who revised the text.

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REVIEW ARTICLE
Year : 2011  |  Volume : 15  |  Issue : 4  |  Page : 310-
 
317
Priti Basgauda Patil1, Basgauda Ramesh Patil2 .Saliva: A diagnostic biomarker of
periodontal diseases

1
 Department of Periodontology, Tatyasaheb Kore Dental College and Research Centre, Kolhapur, Maharashtra, India
2
 Department of Pediatric and Preventive Dentistry, Dr. D.Y. Patil Dental College and Hospital, Maharashtra, India

Date of Submission 06-Dec-2010

Date of Acceptance 11-Nov-2011
Date of Web Publication 2-Feb-2012

       

Correspondence Address:
Priti Basgauda Patil
Department of Periodontology, Tatyasaheb Kore Dental College and Research Centre, New Pargaon - 417 137,
Kolhapur, Maharashtra 
India

DOI: 10.4103/0972-124X.92560

PMID: 22368352

   Abstract  

Early detection of disease plays a crucial role in successful therapy. Early diagnosis and management reduces
the severity and possible complications of the disease process. To overcome this challenge, medical researchers
are devoted to finding molecular disease biomarkers that reveal a hidden lethal threat before the disease
becomes complicated. Saliva, an important physiologic fluid, containing a highly complex mixture of substances,
is rapidly gaining popularity as a diagnostic tool. Periodontal disease is a chronic disease of the oral cavity
comprising a group of inflammatory conditions affecting the supporting structures of the dentition. In the field of
periodontology, traditional clinical criteria are often insufficient for determining sites of active disease, for
monitoring the response to therapy, or for measuring the degree of susceptibility to future disease progression.
Saliva, as a mirror of oral and systemic health, is a valuable source for clinically relevant information because it
contains biomarkers specific for the unique physiologic aspects of periodontal diseases. This review highlights
the various potentials of saliva as a diagnostic biomarker for periodontal diseases.

Keywords: Biomarker, diagnostic fluid, periodontal diseases, saliva

How to cite this article:

Patil PB, Patil BR. Saliva: A diagnostic biomarker of periodontal diseases. J Indian Soc Periodontol 2011;15:310-
7

How to cite this URL:

Patil PB, Patil BR. Saliva: A diagnostic biomarker of periodontal diseases. J Indian Soc Periodontol [serial online]
2011 [cited 2013 Jul 17];15:310-7. Available from: http://www.jisponline.com/text.asp?2011/15/4/310/92560

   Introduction  

Early detection of disease plays a crucial role in successful therapy. In most cases, the earlier the disease is
diagnosed, the more likely it is to be successfully cured or well controlled. However, most systemic diseases are
not diagnosed until morbid symptoms become apparent in the late phase. To overcome this challenge, medical
researchers are devoted to finding molecular disease biomarkers that reveal a hidden lethal threat before the
disease becomes complicated. [1]

   Need for a New Biomarker  

Currently, three major limitations have prevented people from recognizing the full potential of disease detection,
and have seriously hampered the development of clinical diagnostics, namely:

1. Lack of definitive molecular biomarkers for specific diseases;

2. Lack of an easy and inexpensive sampling method with minimal discomfort; and
3. Lack of an accurate, easy-to-use, and portable platform to facilitate early disease detection.

Saliva, an oral fluid that contains an abundance of proteins and genetic molecules and is readily accessible via a
totally noninvasive approach, has long been recognized as the potential solution to these limitations. [1] 

Saliva provides an easily available, noninvasive diagnostic medium for a rapidly widening range of diseases and
clinical situations. [2]

In the field of periodontology, traditional clinical criteria are often insufficient for determining sites of active
disease, for monitoring the response to therapy or for measuring the degree of susceptibility to future disease
progression. Saliva as a mirror of oral and systemic health is a valuable source for clinically relevant information
because it contains biomarkers specific for the unique physiological aspects of periodontal diseases.

   Periodontal Disease  

Periodontal disease is a chronic disease of the oral cavity comprising a group of inflammatory conditions
,
affecting the supporting structures of the dentition. [3] [4] The natural history of periodontitis follows a discontinuous
,
pattern of exacerbation and remission characterized by disease activity and inactivity. [5] [6] Periodontitis is a
multifactorial disease which is affected by both genetic and environmental factors. [7] 

Clinical parameters such as probing depth, attachment level, bleeding on probing, plaque index, and radiographic
assessment of alveolar bone loss provide information on the severity of periodontitis but they do not measure
disease activity, whereas microbiological tests, analysis of host response, and genetic analyses have been
,
proposed in an effort to monitor and identify patients at increased risk for periodontitis. [6] [8]

   Saliva as a Diagnostic Fluid in Periodontal Diseases  

Proposed salivary diagnostic markers for periodontal diseases have included serum and salivary molecules such
as immunoglobulins, enzymes constituents of gingival crevicular fluid, bacterial components or products, volatile
,
compounds, and phenotypic markers, such as epithelial keratins. [2] [8] 
    Collection of Saliva  

The fluid mostly collected for salivary diagnostic purpose is expectorated whole saliva, a mix composed largely of
the secretions from the major salivary glands along with the modest contributions from the minor salivary glands
and gingival crevicular fluid.

Unstimulated or resting saliva is usually collected by passive drooling into a graduated tube or preweighed vial so
that flow rate per unit time can be measured. [2]

When volume measurement is not required, saliva can be collected on cotton swabs, cotton rolls, gauze or filter
paper strips, then eluted or centrifuged or aspirated directly from the floor of the mouth with plastic pipettes. [2]

When large volumes of saliva are required for analytical purposes, saliva is stimulated by a masticatory or
gustatory stimulus, expectorated and handled in a similar manner as the unstimulated fluid. Softened paraffin
wax or a washed rubber band are the usual masticatory stimuli and 2% citric acid applied directly to the tongue is
the standard gustatory stimulus. [2]

Many a times secretions from individual glands are preferred and this can be accomplished in a noninvasive
manner with suitable collecting devices. Parotid saliva is best collected with plastic modifications of a single cup
first introduced by Carlson and Crittenden in 1910. Now disposable and individualized collectors have been
introduced for this purpose. Submandibular-sublingual saliva can be collected by customization of a basic plastic
collector or by aspiration from the duct openings with micropipettes [Figure 1]. [2]

Figure 1: Biomarkers seen in saliva

Click here to view

   Salivary Markers of Periodontal Diseases  

The various salivary biomarkers are as follows.

Markers affecting the dental biofilm

Specific markers

Immunoglobulins (Ig) are important specific defense factors of saliva. The predominant immunoglobulin in saliva
is secretory IgA (sIgA), which is derived from plasma cells in the salivary glands. Lesser amount of IgG and IgM
are also found in saliva. IgA, IgG, and IgM influence the oral microbiota by interfering with the bacterial
,
adherence or by inhibiting bacterial metabolism. [3] [9] There are two subclasses of IgA: IgA1 and IgA2. IgA1 is
predominant in serum while IgA2 is found in higher concentrations in external secretions, that is, tears, saliva,
and milk. [10]

Many studies have been attempted to determine a relationship between salivary levels of sIgA and various forms
of periodontal diseases. It was found that there was a positive correlation between the severity of inflammation
,
and IgA concentration. [11] [12]

Specific immunoglobulins in saliva directed toward periodontal pathogens have also been examined for their
diagnostic potential. Eggert et al. [13] reported that saliva from treated periodontitis patients had higher IgA and
IgG levels to periodontal pathogens porphyromonas. gingivalis and Treponema. denticola than as compared to
saliva from control subjects. Sandholm et al. [14] found increased concentrations of salivary IgG
to Aggregatibacter. actinomycetemcomitans in patients of aggressive periodontitis.

Salivary enzymes
Salivary enzymes can be produced by salivary glands, oral microorganisms, polymorphonuclear leukocytes, oral
epithelial cells derived from gingival crevicular fluid (GCF).

Lysozyme is an antimicrobial enzyme with the ability to cleave chemical bonds in the bacterial cell wall. It can
lyse some bacterial species by hydrolyzing glycosidic linkages in the cell wall peptidoglycan. It may also cause
lysis of bacterial cells by interacting with monovalent anions and with proteases found in saliva. This combination
leads to destabilization of the cell membrane, probably as a result of the activation and deregulation of
endogenous bacterial autolysins. Patients with low levels of lysozyme in saliva are more susceptible to plaque
accumulation, which is considered a risk factor for periodontal disease. [15]

Peroxidase is a salivary enzyme produced by acinar cells in the salivary glands. This enzyme removes toxic
hydrogen peroxide produced by oral microorganisms and reduces acid production in the dental biofilm, thereby
decreasing plaque accumulation and the establishment of gingivitis and caries. Patients with periodontal disease
have demonstrated high levels of this enzyme in saliva. [16]

Salivary ions

Calcium (Ca) is the ion that has been most intensely studied as a potential marker for periodontal disease in
saliva. Sewon et al. [17]reported a higher concentration of Ca detected in whole stimulated saliva from the
periodontitis patients. The authors concluded that an elevated Ca concentration in saliva was characteristic of
patients with periodontitis.

Nevertheless, the importance of the salivary Ca concentration in relationship to progression of periodontal

disease is not defined. Considering the distribution of Ca, this ion appears to hold only limited promise as a
marker for periodontal disease. [8]

Nonspecific markers

proteins: A number of studies have examined the correlation between nonenzymatic, nonimmunoglobulin
proteins in saliva and periodontal disease. 

Mucins are glycoproteins produced by submandibular and sublingual salivary glands and numerous minor
salivary glands. The physiological functions of the mucins (MG1 and MG2) are cytoprotection, lubrication,
protection against dehydration, and maintenance of viscoelasticity in secretions. The mucin, MG2, affects the
aggregation and adherence of bacteria and is known to interact with A. actinomycetemcomitans, and a
decreased concentration of MG2 in saliva may increase colonization with this periodontopathogen. [3] 

Lactoferrin is an iron-binding glycoprotein produced by salivary glands, which inhibits microbial growth by
sequestering iron from the environment, thus depriving bacteria of this essential element. Lactoferrin is strongly
upregulated in mucosal secretions during gingival inflammation and is detected at a high concentration in saliva
of patients with periodontal disease compared with healthy patients. [18] 

Histatin is a salivary protein with antimicrobial properties and is secreted from parotid and submandibular glands.
It neutralizes the endotoxic lipopolysaccharides located in the membrane of gram-negative bacteria. Histatin is
also an inhibitor of host and bacterial enzymes involved in the destruction of the periodontium. In addition to its
antimicrobial activities, histatin is involved in the inhibition of the release of histamine from mast cells, affecting
,
their role in oral inflammation. [3 19]

Fibronectin is a glycoprotein that promotes selective adhesion and colonization of certain bacterial species while
inhibiting others. It mediates adhesion between cells and is also involved in chemotaxis, migration, inflammation,
and wound healing and tissue repair. [8] 

Cystatins (cysteine proteinases) are proteolytic enzymes originated from pathogenic bacteria, inflammatory cells,
osteoclasts, and fibroblasts. These enzymes have collagenolytic activity, which may cause tissue
destruction. [8] Cystatins are physiological inhibitors of cysteine proteinases, and may function by modulating
enzyme activity in the periodontium.

Cystatins are present in a variety of tissues and body fluids, including saliva. Cystatins were found in saliva
collected from the submandibular and sublingual salivary glands, and to a lesser degree in saliva from the parotid
gland. [20]

Platelet activating factor (PAF) is a potent phospholipid mediator of inflammation. Garito et al. found a positive
correlation between PAF and periodontal inflammation. [21] Various other studies also showed similar findings but
none of the authors discussed the potential diagnostic significance of their findings. [8]

Amino acids Several studies have examined the levels of free amino acids in saliva in relation to periodontal
status. It appears that in some patients, elevated levels of certain amino acids, especially proline, may be
 ,
detected. [22] [23] These amino acids probably appear in whole saliva as a result of bacterial metabolism or
degradation of salivary proteins rich in proline. In another study, by the same investigators, no differences in
amino acid concentrations in saliva were found between patients with progressive periodontitis and controls. The
authors concluded that levels of amino acids in oral fluids (including GCF) has no diagnostic significance for
periodontal disease. [24]

Growth factors

growth factor (EGF) is involved in oral wound healing and functions with hormone-like properties to stimulate
epithelial cells. In humans, the parotid gland is the major source of EGF. [8]

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor or vasculotropin, is a
multifunctional angiogenic cytokine important in inflammation and wound healing. This cytokine was found to be
a component of whole saliva. Higher levels of VEGF were detected in whole saliva from periodontitis patients. [8] 

Epithelial keratins

Epithelial cells from the lining of the oral cavity are found in saliva, but the contribution of crevicular or pocket
epithelial cells to the total number of salivary epithelial cells is not known. [25] Furthermore, detection of keratins by
monoclonal antibodies may have diagnostic value in the detection of epithelial dysplasia, oral cancer,
odontogenic cysts, and tumors. [26] 

It has been suggested that phenotypic markers for junctional and oral sulcular epithelia might eventually be used
as indicators of periodontal disease, however, there are no studies that demonstrate an association between
number or type of epithelial cells or specific types of keratins in saliva and progression of periodontitis. [8]

Hormones

, ,
Cortisol: Recent studies have suggested that emotional stress is a risk factor for periodontitis. [7] [27] [28] One
mechanism proposed to account for the relationship is that elevated serum cortisol levels associated with
emotional stress exert a strong inhibitory effect on the inflammatory process and immune response. [29] The
presence of cortisol in saliva has been recognized for more than 40 years. [30]Recently, salivary cortisol levels
were used to evaluate the role of emotional stress in periodontal disease. Higher salivary cortisol levels were
detected in individuals exhibiting severe periodontitis, a high level of financial strain, and high emotion focused
coping, as compared to individuals with little or no periodontal disease, low financial strain, and low levels of
emotion-focused coping. [31] While it appears that emotional stress, as reflected by salivary cortisol levels, is a risk
factor for periodontal disease, attempts to diagnose periodontal disease severity or activity based on salivary
cortisol levels are premature. It can be argued that in addition to changes in the host response (ie, smoking, poor
oral hygiene, and poor compliance), stress also leads to behavioral changes, which could have a significant
effect on the periodontium. Additional studies are required to determine the diagnostic value of salivary cortisol
for periodontal disease. [8]

Inflammatory cells

The number of leukocytes in saliva varies from person to person, and the cell counts vary for an individual during
the course of the day. The majority of salivary leukocytes enter the oral cavity via the gingival
crevice. [32] Klinkhammer [33] standardized collection and counting of leukocytes in saliva and developed the
orogranulocytic migratory rate (OMR). Raeste et al., [34] in their study, indicated that the OMR reflects the
presence of oral inflammation, and suggested that this measure can be used as a laboratory test. 

Occult blood in saliva in relation to gingival inflammation has also been examined using a home-screening
test. [35] 

Bacteria

Specific species of bacteria colonizing the subgingival environment have been implicated in the pathogenesis of
, ,
periodontal disease. [36] [37] [38] It has been suggested that microorganisms in dental plaque can survive in saliva,
and can utilize salivary components as a substrate. It was shown that saliva could serve as a growth medium for
oral Streptococus species and Actinomyces. viscous. [39] 

In a study by De Jong et al., [40] microorganisms from supragingival plaque were grown on saliva agar. When
supragingival plaque was plated on saliva and blood agar plates, the composition of the microflora isolated from
the plates were similar. The authors concluded that the supragingival microflora could utilize saliva as a complete
nutrient source. Studies have also shown the presence of periodontopathic microorganisms, A.
,
actinomycetemcomitans, P. gingivalis, Prevotella intermedia, and T. denticola in whole saliva. [41] [42] 
An oral microbial rinse test (Oratest) was described by Rosenberg et al. [43] In this study Oratest was found to be
a simple method for estimating oral microbial levels. In a companion study, Oratest results were correlated with
plaque index and gingival index scores, and the authors stated that this test provides a reliable estimate of
gingival inflammation. [44]

Volatiles

Volatile sulfur compounds, primarily hydrogen sulfide and methylmercaptan, are associated with oral malodor.
Salivary volatiles have been suggested as possible diagnostic markers and contributory factors in periodontal
disease.

In a study of self-estimation of oral malodor, estimation of malodor based on saliva yielded a significant
correlation with objective parameters.[45] 

Markers in saliva via GCF

As GCF traverses through inflamed periodontal tissues en route to the sulcus, biological molecular markers are
gathered from the surrounding areas and are subsequently eluted into whole saliva.

Markers of periodontal soft tissue inflammation

Proinflammatory cytokines, such as prostaglandin E2 (PGE2), interleukin (IL)-1beta, IL-6, and tumor necrosis
factor-alpha are released from cells of the junctional epithelium, connective tissue fibroblasts, macrophages, and
polymorphonuclear leukocytes. Enzymes, such as matrix metalloproteinase (MMP)-8, MMP-9, and MMP-13,
produced by polymorphonuclear leukocytes and osteoclasts, all lead to the degradation of connective tissue
collagen and alveolar bone. Studies have shown that PGE2 acts as a potent vasodilator and increases capillary
permeability, which elicits clinical signs of redness and edema. It also stimulates fibroblasts and osteoclasts to
increase the production of MMPs. [46] 

Markers of alveolar bone loss

Many different biomarkers associated with bone formation, resorption, and turnover, such as alkaline
phosphatase, osteocalcin, osteonectin, and collagen telopeptidases, have been evaluated in GCF and
saliva. [47] These mediators are associated with local bone metabolism as well as with systemic conditions.

Matrix metalloproteinases (MMP): They are host proteinases responsible for both tissue degradation and
remodeling. During progressive periodontal breakdown, gingival and periodontal ligament collagens are cleaved
by host cell-derived interstitial collagenases.

MMP-8: It is the most prevalent MMP found in diseased periodontal tissue and GCF. Recently, the level of MMP-
8 was demonstrated to be highly elevated in saliva from patients with periodontal disease using a rapid point-of-
care microfluidic device. [48] Studies are required to evaluate MMP-8, either alone or in conjunction with other
molecular biomarkers, to predict the risk of future disease occurrence and to monitor treatment interventions.

Gelatinase (MMP-9): Another member of the collagenase family, is produced by neutrophils and degrades
collagen intercellular ground substance. In their study, Teng et al. [49] found a twofold increase in the mean MMP-
9 levels in patients with progressive attachment loss. Given these results, future use of MMP-9 in oral fluid
diagnostics may serve as a guide in periodontal treatment monitoring.

Collagenase-3: Also referred to as MMP-13, is another collagenolytic MMP with an exceptionally wide substrate
specificity. In the future, MMP-13 may be useful for diagnosing and monitoring the course of periodontal disease
as well as for tracking the efficacy of therapy. [50] 

Telopeptide: Given the specificity and sensitivity for bone resorption, pyridinoline cross-links, such as pyridinoline
cross-linked carboxyterminal telopeptide of type I collagen represent a potentially valuable diagnostic aid for
periodontal disease. Several investigations have explored the ability of pyridinoline cross-links to detect bone
,
resorption in periodontitis as well as in response to periodontal therapy.[51] [52] These studies assessing the role of
GCF carboxyterminal telopeptide of type I collagen levels as a diagnostic marker of periodontal disease activity
have produced promising results to date. Carboxyterminal telopeptide of type I collagen has been shown to be a
promising predictor of both future alveolar bone and attachment loss.

Controlled human longitudinal trials are needed to establish fully the role of salivary carboxyterminal telopeptide
of type I collagen as a predictor of periodontal tissue destruction, disease activity and response to therapy in
periodontal patients.

Osteocalcin: Elevated serum osteocalcin levels have been found during periods of rapid bone turnover, such as
in osteoporosis and multiple myeloma and during fracture repair. Therefore, studies have investigated the
relationship between GCF osteocalcin levels and periodontal disease.

When a combination of the biochemical markers osteocalcin, collagenase, prostaglandin E2, alpha-2
macroglobulin, elastase, and alkaline phosphatase was evaluated, increased diagnostic sensitivity and specificity
values of 80% and 91%, respectively, were reported. [53] 

Osteopontin: It is a single-chain polypeptide with a molecular weight of approximately 32,600. In bone matrix,
osteopontin is highly concentrated at sites where osteoclasts are attached to the underlying mineral surface (ie,
the clear zone attachment areas of the plasma membrane). Results from periodontal studies indicated that
osteopontin concentrations in GCF increased proportionally with the progression of disease; and when
nonsurgical periodontal treatment was provided, the osteopontin levels in GCF were significantly reduced.
Although additional long-term prospective studies are needed, at this point osteopontin appears to hold promise
as a possible salivary biomarker of periodontal disease progression. [54] 

Systemic markers

C-reactive protein is produced by the liver and is stimulated by circulating cytokines, such as tumor necrosis
factor-alpha and interleukin-1, from local and⁄or systemic inflammation such as periodontal inflammation.
Circulating C-reactive protein may reach saliva via GCF or the salivary glands. High levels of C-reactive protein
have been associated with chronic and aggressive periodontal diseases and with other inflammatory
biomarkers. [19] C-reactive protein has recently been shown to be measurable in saliva from periodontal patients
using a lab-on-a-chip method. [3]

Oxidative stress marker

Oxidative stress is defined as the result of an imbalance between oxidant factors and protective antioxidant
systems; it may occur due to an excess of free radicals, or by the diminishing of the antioxidant systems.
Oxidative stress is enhanced during periodontitis. [55] Oxidative stress can result in DNA damage, including
oxidation of nucleosides. 8-Hydroxydeoxyguanosine (8-OHdG) is an oxidized nucleoside that is excreted in the
bodily fluids with DNA repair. It has been demonstrated that the 8-OHdG in bodily fluids can act as a biomarker of
oxidative stress. [56] 

Studies have shown that saliva is a biological product that can be easily collected and may be used for the
quantification of 8-OHdG as an oxidative stress biomarker in the diagnosis and monitorization of the treatment in
,
periodontitis. [57] [58] 

Antioxidative capacity of saliva

Physiologically free radical/reactive oxygen species in the mouth are derived mainly from polymorphonuclear
neutrophils (PMN), which may also help to control bacterial growth by the well-known ''respiratory burst'' (RB).
Such physiological processes are usually efficiently counteracted by intrinsic antioxidant systems: if such
systems fail, tissue damage can result. [59] 

Saliva may constitute a first line of defence against free radical-mediated oxidative stress, since the process of
mastication promotes a variety of such reactions, including lipid peroxidation. [60] Moreover, during gingival
inflammation, gingival crevicular fluid (GCF) flow increases, adding to saliva with products from the inflammatory
response. This is why the antioxidant capacity of saliva is of increasing interest.

Saliva is rich in antioxidants, mainly uric acid, with lesser contributions from albumin, ascorbate and
glutathione. [61] It has been reported that uric acid is the major antioxidant in saliva, accounting for more than 85%
of the total antioxidant activity of resting and stimulated saliva from both healthy and periodontally compromised
subjects. [61] 

Antioxidants such as albumin, ascorbic acid, glutathione and traces of transferrin, lactoferrin, and caeruloplasmin
are also found in saliva. [59]

Emerging salivary diagnostic tools

The use of saliva for translational and clinical application has emerged at the forefront. Most relevant to
periodontal diseases are the emerging toolboxes of the salivary proteome and the salivary transcriptome for early
detection, disease progression, and therapeutic monitoring. Using these emerging technologies, it has been
,
shown that salivary proteins and RNAs can be used to detect oral cancer [62] [63]and Sjogren's syndrome. [64] The
stage is now poised to use these technologies for translational and clinical applications in periodontal diseases.

Salivary proteome: The proteome is the protein complement of the genome, and proteomics is analysis of the
portion of the genome that is expressed. The proteomes in body fluids are valuable due to their high clinical
potential as sources of disease markers. In principle, a global analysis of the human salivary proteomes can
provide a comprehensive spectrum of oral and general health. Furthermore, analysis of salivary proteomes over
the course of complications may unveil morbidity signatures in the early stage and monitor disease progression.
Significant progress has been made in cataloguing human saliva proteins and exploring their post-translational
modifications. By using both two-dimensional gel electrophoresis⁄mass spectrometry and "shotgun" proteomics
,
approaches, Hu et al. [64] [65] identified 309 distinct proteins in human whole saliva.

Collectively, 1166 salivary proteins have been identified: 914 from the parotid fluid and 917 from the combined
submandibular and sublingual fluids by Denny et al. [66]

Salivary transcriptome: Salivary transcriptome is an emerging concept. In addition to salivary proteome, salivary

transcriptomes (RNA molecules) were discovered that are unusually stable in saliva. [67] They included mRNA
molecules that cells use to convey the instructions carried by DNA for subsequent protein production. This
discovery presented a second diagnostic alphabet in saliva and opened a door to another avenue of salivary
transcriptomic diagnostics. [1]

Li et al. [67] found that RNA molecules elevated in oral cancer tissues are also elevated in saliva, which prompted
them to examine the scope and complexity of RNA present in human saliva. Other research groups, particularly
from forensic sciences, are focusing on multiplex mRNA profiling for the identification of body fluids, including
,
saliva. [68] [69] 

Possible testing for HIV

To expedite screening and accurately diagnose HIV infection, rapid point-of-care HIV tests have been developed.
Majority of these rapid tests utilize whole blood, plasma, and finger stick blood specimens, whereas a few tests
,
employ nonconventional specimens, such as saliva, oral mucosal fluid and urine. [70] [71] Although salivary and oral
fluid-based tests have been in development for the past 20 years, it is only recently that oral fluid-based, rapid
point-of-care tests have increased in popularity. [71] Oral fluid tests are based on a salivary component, the oral
mucosal transudate or crevicular fluid, an interstitial transudate rich in IgG antibodies, used for diagnosing HIV
, ,
infection. [70] [71] [72] Although accuracy is very high, oral fluid test results are considered preliminary. Therefore, the
results of oral fluid tests require confirmatory testing with conventional tests, such as ELISA and/or western
blot. [73] 

   Discussion  

In the field of periodontal diagnosis, there has been a steady growing trend during the last 2 decades to develop
tools to monitor periodontitis. Currently, diagnosis of periodontal disease relies primarily on clinical and
radiographic parameters. These measures are useful in detecting evidence of past disease, or verifying
periodontal health, but provide only limited information about patients and sites at risk for future periodontal
breakdown. Numerous markers in saliva have been proposed and used as diagnostic tests for periodontal
disease.

Ideally, diagnostic tests should demonstrate high specificity and sensitivity. Given the complex nature of
periodontal disease, it is unlikely that a single marker will prove to be both sensitive and specific. A combination
of two or more markers may provide a more accurate assessment of the periodontal patient.

Interest in saliva as a diagnostic medium is escalating due to its many advantages over other diagnostic biofluids.
Both saliva and blood serum contain similar proteins and RNA, which is why saliva is considered a "mirror to the
"
body. [74] Saliva is readily available which makes the collection process fairly straightforward, even when multiple
samples are needed. Its collection is noninvasive, which makes the procedure more acceptable to patients and
more conducive to a stress-free appointment. Many of the hazards associated with blood collection do not apply
to saliva. There is no potential for cross-contamination among patients when used improperly and present a
danger to health care personnel. Because of the low concentrations of antigens in saliva, HIV, and hepatitis
infections are much less of a danger from saliva than from blood. [75] Saliva is also easier to handle because it
does not clot. As salivary testing becomes more commonplace, the costs could drop below those currently
incurred for urine/blood sampling. However, due to the uniqueness of the technique, the analysis today is still
quite expensive.

GCF has several diagnostic advantages, such as contributing inflammatory mediators and tissue-destructive
molecules associated with periodontitis that appear, and can be detected, in GCF. However, GCF analyses are
time consuming, requiring multiple sampling of individual tooth sites. The procedure is labor intensive and
somewhat technically demanding, requiring equipment for calibrating and measuring fluid volumes. Finally,
analysis is expensive, requires laboratory-based assays and generally cannot be done chairside. In addition,
GCF analyses involve miniscule amounts of fluid, often approximately 1 μL, which has an impact on laboratory
analysis, and can be contaminated with blood, saliva, or plaque. [76] Given some of the problems inherent in
sampling GCF, the analysis of salivary biomarkers offers some advantages. Acquisition of saliva is easy,
noninvasive, rapid, and requires less manpower and materials than GCF. However, in contrast with GCF, whole
, ,
saliva clearly provides different diagnostic information. [77] [78] [79] 

Because of the simple and noninvasive method of collection, salivary diagnostic tests appear to hold promise for
the future.

Novel technologies such as lab-on-a-chip and microfluidic devices have the potential to manage complex oral
fluids, such as saliva and GCF, and to provide a determination of a patient's periodontal disease-risk profile,
current disease activity, and response to therapeutic interventions. This approach should accelerate clinical
decision-making and monitoring of episodic disease progression in a chronic infectious disease such as
periodontitis. [8]

   Conclusion  

Saliva, like blood, contains an abundance of protein and nucleic acid molecules that reflects physiological status;
however, unlike other bodily fluids, salivary diagnostics offer an easy, inexpensive, safe, and noninvasive
approach for disease detection, and possess a high potential to revolutionize the next generation of diagnostics.

Although challenges remain ahead, the use of saliva-based oral fluid diagnostics appear promising for future
application to diagnose periodontal diseases and to prognosticate periodontal treatment outcomes.

   Acknowledgments  

The authors would like to thank Dr. Hari Menon, Dr. Abhijit Gurav, and Dr. Abhijeet Shete for thoughtful
discussions, and assistance in preparation of the manuscript.

 
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Caffesse RG, Nasjleti CE, Anderson GB, Lopatin DE, Smith BA, Morrison
EC.Periodontal healing following guided tissue regeneration with citric acid and
fibronectin application. J Periodontol. 1991 Jan;62(1):21-9.

Source
Department of Periodontics, University of Texas Health Science Center, Houston.
Abstract
This study was undertaken to determine the effects of guided tissue regeneration (GTR) with and
without citric acid conditioning and autologous fibronectin application. The study subjects were four
female beagle dogs with spontaneous periodontitis. The dogs were given thorough root debridement
and 4 weeks later, mucoperiosteal flaps were raised on both sides of the mandible involving the 2nd,
3rd, and 4th premolar and 1st molar teeth. After debridement, notches were placed on the roots at the
level of supporting bone. Citric acid (pH 1) was topically applied for 3 minutes on the exposed root
surfaces of one side (experimental). The roots were irrigated with normal saline solution. Both the root
surfaces and the inner surface of the flap were then bathed in autologous fibronectin in saline.
Following this, Gore-Tex periodontal material was adapted to the roots of each tooth and sutured. The
contralateral side, serving as control, was treated by surgery and application of Gore-Tex periodontal
material only. All membranes were removed 1 month after surgery, and the dogs sacrificed at 3
months. Both mesio-distal and bucco-lingual microscopic histological sections were evaluated by
descriptive histology, and linear measurements and surface area determination of the furcal tissues
were made. Periodontal healing following the use of GTR procedure resulted in an increase in
connective tissue and alveolar bone regeneration. Adjunctive critic acid plus autologous fibronectin
produced slightly better results, but these differences were not statistically significant for this sample.

PMID:

2002428

[PubMed - indexed for MEDLINE]

William V. Gianobille , Khalaf F. Al-Shammari, David P. Sarment. Matrix Molecules

And Growth Factors As Indicators Of Periodontal Disease Activity. Periodontology
2000, Vol. 31, 2003, 125–134 Copyright C Blackwell Munksgaard 2003

Printed in Denmark. All rights reserved PERIODONTOLOGY 2000

ISSN 0906-6713
In general, chondroitin-4-sulfate is the most common
glycosaminoglycan in the periodontium but
different distributions exist. For example, core proteins
of lower molecular weight are characteristic of
bone (34). In this tissue as well as in cementum,
dermatan sulfate is rare (104), but it is more common
in the periodontal ligament and the gingiva (6),
probably reflecting a functional involvement of the
molecule in the mineralization process
Biochemical Markers of Bone Turnover Part I: Biochemistry and
Variability
Markus J Seibel

Author information ► Copyright and License information ►

This article has been cited by other articles in PMC.

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Abstract

With the ageing population in most countries, disorders of bone and mineral metabolism
are becoming increasingly relevant to every day clinical practice. Consequently, the
interest in, and the need for effective measures to be used in the screening, diagnosis and
follow-up of such pathologies has markedly grown. Together with clinical and imaging
techniques, biochemical tests play an important role in the assessment and differential
diagnosis of metabolic bone disease.

In recent years, the isolation and characterisation of cellular and extracellular

components of the skeletal matrix have resulted in the development of molecular
markers that are considered to reflect either bone formation or bone resorption. These
biochemical indices are non-invasive, comparatively inexpensive and, when applied and
interpreted correctly, helpful tools in the diagnostic and therapeutic assessment of
metabolic bone disease.

Part I of this article provides an overview of the basic biochemistry of bone markers, and
sources of non-specific variability. Part II (to be published in a subsequent issue of this
journal) will review the current evidence regarding the clinical use of biochemical
markers of bone remodelling in metabolic and metastatic bone disease.

Go to:

Introduction

Bone is a metabolically active tissue that undergoes continuous remodelling by two

counteracting processes, namely bone formation and bone resorption. These processes
rely on the activity of osteoclasts (resorption), osteoblasts (formation) and osteocytes
(maintenance). Under normal conditions, bone resorption and formation are tightly
coupled to each other, so that the amount of bone removed is always equal to the amount
of newly formed bone (Figure 1). This balance is achieved and regulated through the
action of various systemic hormones (e.g. PTH, vitamin D, other steroid hormones) and
local mediators (e.g. cytokines, growth factors). In contrast, somatic growth, ageing,
metabolic bone diseases, states of increased or decreased mobility, therapeutic
interventions and many other conditions are characterised by more or less pronounced
imbalances in bone turnover. The results of such uncoupling in bone turnover are often
changes in bone structure, strength and mass. While bone structure and strength are
difficult to measure in vivo, bone mass can be assessed by densitometric techniques. In
contrast to these rather static measures, however, molecular markers of bone
metabolism are helpful tools to detect the dynamics of the metabolic imbalance itself. 1,2

Figure 1

The bone remodelling cycle. Under normal conditions, the resorption (osteoclast) phase
takes approximately 10 days, which is then followed by a formation (osteoblast) phase
that can last for up to 3 months.

Although the currently available markers of bone turnover include both enzymes and
non-enzymatic peptides derived from cellular and non-cellular compartments of bone,
they are usually classified according to the metabolic process they are considered to
reflect. Most biochemical indices of bone resorption are related to collagen breakdown
products such as hydroxyproline or the various collagen cross-links and telopeptides.
Other markers of bone resorption include non-collagenous matrix proteins such as bone
sialoprotein (BSP), or osteoclast-specific enzymes like tartrate-resistant acid
phosphatase or cathepsin K. In contrast, markers of bone formation are either by-
products of collagen neosynthesis (e.g. propeptides of type I collagen), or osteoblast-
related proteins such as osteocalcin (OC) and alkaline phosphatase (AP). For clinical
purposes, therefore, markers of bone formation are distinguished from indices of bone
resorption (Table 1, Figure 2). This distinction, however, is not as sharp as it may appear.
For example, some marker components reflect, at least in part, both bone formation and
bone resorption (e.g. hydroxyproline, certain OC fragments). Furthermore, most of the
molecules used as markers of bone turnover are also present in tissues other than bone,
and non-skeletal processes may therefore influence their circulating or urinary levels.
Finally, changes in markers of bone turnover are not disease specific but reflect, as an
integral measure, alterations in the metabolism of the entire skeletal envelope
independently of the underlying cause. Hence, results of bone marker measurements
should always be interpreted against the background of their basic science and the
clinical picture.

Figure 2

Biochemical markers of bone remodelling.

Table 1

Markers of bone turnover.

Go to:

Markers of Bone Formation

Bone formation markers are products of active osteoblasts expressed during different
phases of osteoblast development. They are considered to reflect different aspects of
osteoblast function and of bone formation. All markers of bone formation are measured
in serum or plasma.

Serum Total Alkaline Phosphatase (AP)

AP is a ubiquitous, membrane-bound tetrameric enzyme attached to glycosyl-

phosphatidylinositol moieties located on the outer cell surface. 3 The precise function of
the enzyme is yet unknown, but it obviously plays an important role in osteoid formation
and mineralisation.4 The total AP serum pool (TAP) consists of several dimeric isoforms,
which originate from various tissues: liver, bone, intestine, spleen, kidney, and placenta.
In addition, certain tumours may express macromolecular forms of AP (e.g. “Nagao
AP”).5,6

The physiological isoforms of AP are coded by four gene loci, including three tissue-
specific and one non-tissue-specific gene on chromosome 1. The latter encodes for the
most abundant isoforms, namely bone, liver, and kidney AP. The differences between
these non-specifically encoded isoenzymes are solely due to post-translational
modifications in the carbohydrate moiety.7 In adults with normal liver function,
approximately 50% of the total AP activity in serum is derived from the liver, whereas
50% arises from bone.8 In children and adolescents the bone-specific isoenzyme
predominates (up to 90%) because of skeletal growth. 9,10

Many techniques have been developed to differentiate between the two main isoforms of
circulating AP, including heat denaturation, electrophoresis, precipitation, selective
inhibition and, more recently, immunoassays.11–15 In healthy adults, most methods show a
good correlation between bone specific and total AP (Figure 3). The newer
immunoassays allow simple and rapid quantitation of either enzyme activity or enzyme
mass. However, like all of the other techniques, even these assays show a certain degree
of cross-reactivity between bone and liver AP (15–20%). Therefore, in subjects with high
liver AP, results of bone AP measurements may be artificially high, leading to false
positive results.7, 16, 17

Figure 3

Correlation between serum total and bone specific alkaline phosphatase. Patients with
non-skeletal disease had chronic hepatic failure, chronic obstructive pulmonary disease,
or chronic renal failure. Patients with skeletal disease had Paget’s ...

Serum total AP is the most widely used marker of bone metabolism due to the wide
availability of inexpensive and simple methods. Once liver disease is ruled out, serum
levels of total AP provide a good impression of the extent of new bone formation and
osteoblast activity.18,19 From a clinical perspective, however, detection of the bone-specific
AP (BAP) isoenzyme is increasingly preferred because of its higher specificity. 8, 20, 21

Osteocalcin (OC)

OC is a 5.8 kDa, hydroxyapatite-binding, protein exclusively synthesised by osteoblast,

odontoblasts and hypertrophic chondrocytes.22–24 One of the major characteristics of OC
is 3 vitamin-K dependent, gamma-carboxyglutamic acid (Gla) residues, which are
responsible for the calcium binding properties of the protein. 25 At its carboxy-terminus,
OC can also interact with other proteins, including cell surface receptors. These
functions predispose OC as a molecule active in the organisation of the extracellular
matrix. Earlier research has suggested that OC is involved in the process of osteoid
mineralisation, as the protein is expressed mainly during this phase of bone formation.
However, although OC has been known for more than 20 years, its precise function has
yet to be determined. More recently, new light has been cast on this issue through the
development of an OC knockout mouse model. Unexpectedly, these animals have
increased cortical and trabecular thickness, and their bones seem mechanically more
stable than those of the wild type.26 Although the knockout model awaits further
characterisation, it seems that OC is involved in the bone remodelling process and may
act via a negative feed back mechanism.

OC is considered a specific marker of osteoblast function. 27 It is estimated that, directly

after its release from osteoblasts, the largest part of the newly synthesised protein is
incorporated into the extracellular bone matrix where it constitutes approximately 15%
of the non-collagenous protein fraction. A smaller fraction is released into the circulation
where it can be detected by immunoassays.28–34 Serum levels of immunoreactive OC have
been shown to correlate well with the bone formation rate as assessed by
histomorphometry.35 However, the peptide is readily subject to rapid degradation in
serum, so that both intact peptides and OC fragments of various sizes coexist in the
circulation.36–38 Furthermore, since OC is incorporated into the bone matrix, some
investigators have suggested that OC fragments may be released even during bone
resorption. This may be particularly true for some smaller N-terminal fragments of OC,
which are found in individuals with high bone turnover. 39–41 The ensuing heterogeneity of
OC fragments in serum results in considerable limitations in the clinical application of
this a priori highly specific marker. Thus, the various assays used to measure OC in
serum detect fragments of various sizes and usually, epitope specificity and antibody
cross-reactivity of the assays are ill defined. In practice, different immunoassays have
routinely yielded such varying results in measurements that they are not comparable. 43,45

Two-site immunoassays, utilising antibodies detecting different parts of the OC

molecule, have been introduced that detect the intact 1–49 OC molecule. However, only
one third of the total OC serum pool represents intact OC, and due to the instability of
OC in serum, rapid loss of immunoreactivity is seen with these assays when samples are
left for more than 1 hour at room temperature. To circumvent this problem, newer assays
measure the largest degradation product of OC, the 1–43 (N-terminal/mid-molecule)
fragment. This fragment, which represents one-third of the circulating OC pool, is a
result of proteolytic degradation of the intact molecule and may in part be generated by
active osteoblasts. Although little is know about the function of the N-terminal fragment,
it’s measurement eliminates in part the problem of pre-analytical
instability.45,46 However, quick processing of the blood sample after drawing is essential
for most assays since a loss of reactivity is noted within a few hours at room temperature.
The same applies to sera subjected to multiple thawing, or prolonged storage at
temperatures above −25° C.

Procollagen Type I Propeptides

The procollagen type I propeptides are derived from collagen type I, the most abundant
form of collagen found in bone.45 However, type I collagen is also present in other tissues
such as skin, dentin, cornea, vessels, fibrocartilage, and tendons. In bone, collagen is
synthesised by osteoblasts in the form of pre-procollagen. These precursor molecules are
characterised by short terminal extension-peptides: the amino (N-) terminal propeptide
(PINP) and the carboxy (C-) terminal propeptide (PICP) (Figure 4).46After secretion into
the extracellular space, the globular trimeric propeptides are enzymatically cleaved and
liberated into the circulation.47 PICP has a MW of 115 kDa, is stabilised by disulphide
bonds, cleared by liver endothelial cells via the mannose receptor and therefore has a
short serum half-life of 6–8 minutes.48,49 PINP has a MW of only 70 kDa, is rich in
proline and hydroxyproline and is eliminated from the circulation by liver endothelial
cells by the scavenger receptor. Since both PICP and PINP are generated from newly
synthesised collagen in a stoichiometric fashion, the propeptides are considered
quantitative measures of newly formed type I collagen. Although type I collagen
propeptides may also arise from other sources, most of the non-skeletal tissues exhibit a
slower turnover than bone, and contribute very little to the circulating propeptide pool.

Figure 4

Schematic representation of the collagen type I molecule. The carboxy- and amino-
terminal propeptides are cleaved by specific propeptidases and are partly released into
the circulation. Figure courtesy of Dr Simon Robins, Aberdeen.

Both propeptides are currently measured by specific immunoassays. 50,51 Different studies

have shown good correlations between serum PICP levels and the rate of bone
formation.52,53 While the clinical relevance of PICP in the evaluation of metabolic bone
diseases is still viewed with skepticism,54,55serum PINP appears to be of greater diagnostic
validity. From a practical point of view, the thermostability of the propeptides is an
advantage in that extended transport and storage times are well tolerated without
significant loss of signal. The propeptides share these properties with most of the
parameters of collagen metabolism (e.g., crosslinks, ICTP, NTx, CTx,
hydroxyproline; vide infra.

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Markers of Bone Resorption

Except for tartrate-resistant acid phosphatase the majority of bone resorption markers
are degradation products of bone collagen (Figure 5). More recently, non-collagenous
proteins such as BSP, and osteoclast-derived enzymes such as cathepsin K and L have
been investigated as markers of bone turnover.

Figure 5

Molecular basis of currently used markers of type I collagen-related degradation. For

more details, see text and Table 1. Figure courtesy Dr Simon Robins, Aberdeen.

Hydroxyproline (OHP)

OHP is formed intracellularly from the post-translational hydroxylation of proline and

constitutes 12–14% of the total amino acid content of mature collagen. Ninety percent of
the OHP liberated during the degradation of bone collagen is primarily metabolised in
the liver.56 Subsequently, it is excreted in the urine where it may be detected either as
free or peptide-bound hydroxyproline by colorimetric or HPLC methods. 57,58 Urinary
OHP is usually considered to reflect bone resorption. However, it should be noted that
significant amounts of urinary OHP are derived from the degradation of newly
synthesised collagen.59 In addition, hydroxyproline can be found in other tissues such as
the skin and, moreover, liberated from the metabolism of elastin and C1q. 60,61 Urinary
hydroxyproline is therefore considered an unspecific index of collagen turnover and,
consequently, has been largely replaced by more specific techniques.

Hydroxylysine-Glycosides

The hydroxylysine-glycosides arise during the post-translational phase of collagen

synthesis and occur in two forms, glycosyl-galactosyl-hydroxylysine (GGHL) and
galactosyl-hydroxylysine (GHL) (Figure 5).62 Both components are released into the
circulation during collagen degradation and may be measured in urine by HPLC after
appropriate derivatisation.63 The intrinsic advantage of hydroxylysine over that of
hydroxyproline is that the glycosylated forms are not metabolised and are not influenced
by dietary components.62,63 Moreover, GGHL is present in skin and C1q, while GHL is
more specific for bone. Thus the ratio of GGHL/GHL may allow for the recognition of
existent tissue specificity. Although the hydroxylysines have potential as markers of bone
resorption, their major disadvantage is the current absence of a convenient
immunoassay format.64–66

3-Hydroxypyridinium Crosslinks of Collagen Pyridinoline (PYD) and

Deoxypyridinoline (DPD)

PYD and DPD are formed during the extracellular maturation of fibrillar collagens. As
trifunctional crosslinks, they bridge several collagen peptides and mechanically stabilise
the collagen molecule (Figure 5).67–69 During bone resorption, crosslinked collagens are
proteolytically broken down and the crosslink components are released into the
circulation and the urine.70–72 The measurement of hydroxypyridinium crosslinks is not
influenced by the degradation of newly synthesised collagens and their levels strictly
reflect the degradation of mature i.e. crosslinked collagens. In addition, the urinary
excretion of pyridinium crosslinks is independent of dietary sources since neither PYD
nor DPD are taken up from food.73 Finally, the two hydroxypyridinium components show
a high specificity for skeletal tissues. While PYD is found in cartilage, bone, ligaments
and vessels, DPD is almost exclusively found in bone and dentin. Neither derivative is
present in the collagen of skin or in other sources such as C1q or elastin. 61,74,75 Since bone
has a much higher turnover than cartilage, ligaments, vessels or tendons, the amounts of
PYD and DPD in serum or urine are mainly derived from the skeleton. Thus, the
pyridinium crosslinks are currently viewed the best indices for assessing bone
resorption.76–78

Initially, PYD and DPD were quantified in urine by reverse-phase ion-paired HPLC
technique, combined with a pre-fractionation step using cellulose partition
chromatography, and hydrolysis of urine samples to convert all crosslinks into the
peptide free forms.75,79–81 Currently, automated techniques for the measurement in urine
and serum are available. Analysis of urine by HPLC without initial hydrolysis showed
that 40–50% of the crosslinks were present in peptide-free form. 82 Although the amounts
of free and peptide-bound crosslinks seem to vary with the bone pathology, direct
immunoassays for free and peptide-bound crosslinks are widely used. Under normal
conditions, these assays have been shown to produce results similar to those provided by
the traditional HPLC technique.83–85

Crosslinked Telopeptides of Type I Collagen

The crosslinked telopeptides of type I collagen are derived from specific regions of the
collagen type I molecule, namely the aminoterminal (NTP) and the carboxyterminal
(CTP) telopeptide (Figure 5).

The first collagen telopeptide assay was a RIA for the carboxyterminal type I collagen
telopeptide (ICTP) in serum.86 The respective antibodies were raised against a crosslink-
containing collagen peptide (MW 8.5 kDa) isolated from human bone. The antigenic
determinant requires a trivalent crosslink, including two phenylalanine-rich domains of
the telopeptide region of the alpha-1 chain of type I collagen. Divalently and non-
crosslinked peptides do not react with the antibody, nor do peptides isolated from skin.
Despite the fact that the initial peptide contained pyridinoline, the assay also detected
other crosslink forms such as deoxypyridinoline or pyrrole crosslinks. The ICTP assay
appears to be sensitive for pathological bone resorption as seen in multiple myeloma,
metastatic bone disease and other degradation processes involving hasty breakdown of
skeletal and non-skeletal type I collagen. 87

Another group of immunoassays involve the carboxyterminal telopeptide of type I

collagen, abbreviated CTX (Figure 5).88 Employing a polyclonal antiserum against a
synthetic octapeptide containing the crosslinking site, a first ELISA (termed β-CTX)
recognised the C-terminal type I collagen telopeptide containing an isoaspartyl (=β-
aspartyl) peptide bond in its L-enantiomeric form. 89 The β–L-aspartyl is considered to
result mainly from the ageing of extracellular proteins. Only one peptide strand is
necessary for immunoreactivity. Meanwhile, a monoclonal-based RIA for the non-
isomerised octapeptide (EKAH- αD-GGR) in urine has been developed (“α-
CTX”).90 Simultaneous measurement of both forms may be used to calculate the ratio of
α-CTX/β-CTX as an index of bone turnover. This ratio has been shown to be elevated in
the urine of patients with untreated Paget's disease of bone, where rapid bone formation
and resorption result in an increase of α-CTx (non-isomerised octapeptide). 91

Further to isomerisation, many proteins are also subjected to racemisation of certain

residues. Both processes are considered an effect of ageing, as the extent of racemisation
and isomerisation increases with the time elapsed since synthesis of the protein.
Additionally, antibodies directed against the D-aspartic acid residues in the CTX
molecule have been described.92 Thus, immunoassays now exist (though not
commercially available) for all four possible isomers of the CTX molecule: the native α-L
form, the β-isomerised isoaspartyl peptide (βL), and the respective racemised forms α-D
and β-D. The differential use of these assays may possibly provide information on the
age-dependent changes of collagen in health and disease, although the clinical relevance
is questionable.93
More recently, a sandwich ELISA for the measurement of β-CTX in serum has been
developed. When this assay was described initially, it utilised a polyclonal antiserum
against collagenase-digested collagen type I together with the β-isomerised C-terminal
octapeptide attached to the ELISA plate.94 The newer assay uses two monoclonal
antibodies, and it is claimed that they recognise only dipeptides containing a crosslink
and two β-isomerised peptides with the same sequence as shown for the urinary assay.
Although there is no direct evidence that the antigenic determinant in serum must
contain a crosslink for immunoreactivity, this is indeed highly probable since β-
transformation occurs slower than crosslinking, and single peptides do not react in the
serum CTX-ELISA. However, the crosslink component may be any kind of crosslink,
similar to the ICTP antigen in serum. Presently, standards for the original serum CTX-
ELISA are made from antigens immunopurified from human urine, but automated
assays (Boehringer-Mannheim) utilise synthetic antigens. Serum and urinary CTX values
are highly correlated (r= 0.86) suggesting that the antigen is similar in both analytical
media.

Yet another peptide assay measures the crosslinked N-terminal telopeptide of type I
collagen. This assay (termed NTX-assay) utilises a monoclonal antibody raised against
an epitope on the alpha-2 chain of type I collagen. (Figure 5).95 However, the antibody
seems to react with several crosslinking components, and the presence of a pyridinium
crosslink is not essential for reactivity. As a matter of fact, digests of skin collagen
exhibited similar reactivity with the NTX assay as skeletal extracts. 96 Both the
monoclonal antibody and the assay format are identical for the urine and the serum
based assays. As expected, the two assays show good correlation to each other, and the
analyte seems to be stable at room temperature and during up to three freeze-thaw
cycles.97

Bone Sialoprotein (BSP)

BSP is a phosphorylated glycoprotein with an apparent MW of 70–80 kDa, which

accounts for 5–10% of the non-collagenous matrix of bone. 98,99 The protein has been
shown to be a major synthetic product of active osteoblasts and odontoblasts, but was
also found in osteoclast-like and malignant cell lines. Consequently, BSP or its mRNA is
detected mainly in mineralised tissue such as bone, dentin and at the interface of
calcifying cartilage.100–102 Intact BSP contains an Arg-Gly-Asp (RGD) integrin recognition
sequence, improves the attachment of osteoblasts and osteoclasts to plastic surfaces,
binds preferentially to the α2 chain of collagen, nucleates hydroxyapatite crystal
formation in vitro and appears to enhance osteoclast mediated bone resorption. 103,104 The
protein is therefore considered to play an important role in cell-matrix-adhesion
processes and in the supramolecular organisation of the extracellular matrix of
mineralised tissues.

We and others have developed immunoassays for the measurement of BSP in

serum.105,106 So far, all of these assays are based upon polyclonal antisera, and little is
known about the specific nature of the respective epitopes. Antibodies do not cross-react
with non-collagenous proteins such as osteonectin, fibronectin or osteocalcin. 106 In
serum, the majority of BSP is bound to Factor H, a major regulator of the alternate
complement pathway, which is found at 0.5g/L in serum. Although this phenomenon is
of unknown physiological relevance, BSP/Factor H binding studies suggest that current
immunoassays detect only a fraction of bioavailable BSP in serum. 107 Based upon clinical
data and the rapid reduction of serum BSP levels following intravenous bisphosphonate
treatment, it is assumed that serum BSP reflects processes mainly related to bone
resorption.108

Tartrate-Resistant Acid Phosphatase (TRAP, TRAcP)

This enzyme belongs to the family of the ubiquitous acid phosphatases, of which at least
five different isoforms are known. These isoforms are expressed by different tissues and
cells such as prostate, bone, spleen, platelets, erythrocytes, and macrophages. All acid
phosphatases are inhibited by L(+)-tartrate, except band 5, which was therefore termed
tartrate-resistant acid phosphatase (TRAP or TRAcP). Of the latter, 2 subforms, 5a and
5b are known, and recent research has shown that TRAP-5b is characteristic of
osteoclasts.109 The origin of TRAP-5a is unknown, but may be expressed by macrophages.
The two isoforms 5a and 5b are different in that 5a contains sialic acid, whereas 5b does
not. So far, most assays for the measurement of TRAP in blood have used colorimetry
and detected both isoforms without differentiating between bands 5a and 5b. More
recently, specific immunoassays for TRAP 5b have been described and clinical results
indicate that this marker may be useful to assess osteoclast activity. 110,111The antibodies for
these assays were raised against material isolated from the spleen of a patient with hairy
cell leukemia or against TRAP 5b isolated from human cord plasma. 112,113 For the
conventional TRAP assays, care should be taken after phlebotomy to stabilise the
enzyme since TRAP loses more than 20% of its activity per hour at room temperature.
This can be prevented by the addition of citrate buffer to the sample. 114

Cathepsin K

Cathepsin K is a member of the cysteine protease family that, unlike other cathepsins,
has the unique ability to cleave both helical and telopeptide regions of collagen I. 115,116 Its
clinical relevance was appreciated with the discovery that pycnodysostosis, an autosomal
recessive disease characterised by osteopetrosis, was the result of mutations in the
cathepsin K gene.117 This clinical phenotype has been confirmed in cathepsin K null mice
showing dysfunctional matrix digestion.118,119Immunocytochemical studies have shown
that cathepsin K is located intracellularly in vesicles, granules and vacuoles throughout
the cytoplasm of osteoclasts and that it is secreted into bone resorption lacunae for
extracellular collagen degradation.120 Recently, a new enzyme-linked immunoassay for
measurements of cathepsin K in serum has been developed. Due to the fact that
cathepsin K is expressed and secreted by osteoclasts during active bone resorption,
cathepsin K, and specifically its circulating form, may be a useful and specific
biochemical marker of osteoclastic activity.

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Variability

Most bone turnover markers exhibit significant within-subject variability. This poses a
major problem in the practical use of bone markers: Whenever a change in a bone
marker level is observed in an individual patient (e.g. following an intervention), this
change must be interpreted against the background of the respective marker’s variability.
Therefore, knowledge of the sources of variability and the strategies used to cope with
“background noise” are essential for the meaningful interpretation of bone markers.

In general, non-specific variability comprises both pre-analytical (i.e. mostly subject

related; CVPA) and analytical (i.e. mostly assay related; CV A) factors. Total variability is
considered the sum of pre-analytical and analytical variation and defined as CV T2 =
CVPA2 + CVA2. The ideal marker and assay is characterised by i) excellent analytical
performance (i.e. high precision and accuracy) and ii) minimal and predictable pre-
analytical variability. Unfortunately, no test in clinical chemistry completely meets all of
these criteria. Indeed, most of the currently available assays for biochemical markers of
bone turnover are characterised by substantial pre-analytical variability. The relevant
pre-analytical factors affecting marker variability are summarised in Table 2 and and33.

Table 2

Sources of pre-analytical variability.

Table 3

Pre-analytical and biological characteristics of bone markers.

A number of factors need to be taken into account when employing serial measurements
of biochemical markers to determine changes in bone turnover. To minimise some of the
limitations linked to pre-analytical and analytical variability, standardised sampling and
sample handling are mandatory to obtain reliable results.
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Technical Sources of Variability

In addition to parameters of assay performance (Figure 6 and and7),7), factors such as

the choice of sample (i.e. serum versus urine), mode of sample collection (e.g. 24 hour
collection versus first or second morning void), the appropriate preparation of the
patient (e.g. pre-sampling diet/fasting/exercise before phlebotomy), the correct
handling, processing and storage of specimens should always be considered. This is
important as these technical sources of variability are controllable and hence modifiable.
For the purpose of practical use, some technical aspects of variability are discussed in
more detail in Table 2.

Figure 6

Intra-assay precision profiles for three immunoassays measuring type I collagen

degradation products in urine. A: Deoxypyridinoline (DPD), B: Aminoterminal
crosslinked telopeptide (NTX), C: Carboxyterminal octapeptide (CTX) Reproduced from,
Ju HS, Leung ...

Figure 7

Dilution linearity profiles for three immunoassays measuring type I collagen degradation
products in urine (n=4). A: Deoxypyridinoline (DPD), B: Aminoterminal crosslinked
telopeptide (NTX), C: Carboxyterminal octapeptide (CTX). Reproduced from, ...

Thermodegradation

Some bone markers are sensitive to ambient conditions such as temperature.

Thermodegradation should always be a concern with assays directed against the intact
OC(1–49) molecule. Rapid enzymatic cleavage of the peptide into smaller fragments will
lead to significant signal losses if the serum sample is kept at room temperature for more
than 1–2 hrs. Adding protease inhibitors will delay, but by no means prevent this
process.121 Both the free and conjugated forms of PYD and DPD have been shown to be
stable in urine samples kept at room temperature for several weeks. Several reports show
that pyridinium crosslinks can be stored at –20°C for years and that repeated freeze-
thaw cycles of urine samples have no effect on the concentrations of PYD and DPD. 122

Similar stability has been reported for urinary N-terminal (NTX) and C-terminal (CTX)
collagen type I telopeptides, while ICTP in serum loses up to 12% of the signal when
stored at room temperature for 5 days.123 The stability of glycosylated hydroxylysine
residues has not been fully characterised yet, but it may be necessary to add boric acid to
preserve the urine samples.

The activity of serum tartrate-resistant acid phosphatase (TRAP) declines rapidly during
storage at room temperature or even at −20°C but is stable when stored at −70°C or
lower.114 Multiple freezing-thaw cycles usually have a deleterious effect on serum TRAP
activity. In contrast, serum levels of BSP appear rather stable, both at room temperature,
4°C and −20°C, and have been shown to not change significantly during repeated freeze-
thaw-cycles.124 However, when samples are being exposed to temperatures above 30°C,
an increase in signal is usually seen with the RIA.

Some assays and marker components are sensitive to haemolysis of the sample, resulting
in results that are either too low or too high. This is usually the case for OC and BSP, but
has also been described for TRAP and some other serum markers.

Photolysis

Pyridinium crosslinks in aqueous solutions are unstable when subjected to intensive UV

irradiation.122,123,125 The effect increases with rising pH and has been shown to be greater
for free than for total pyridinoline.122 Urinary NTX and CTX are not affected by UV light
exposure.123

Timing and Mode of Sample Collection

In general, random samples can be used for measurement of most urinary parameters
(but see below for diurnal variation!). For convenience, measurement of bone markers in
urine is usually performed either in the first or second morning void, or in 2 hour
collections. In each case, values need to be corrected for urinary creatinine which
introduces additional pre-analytical and analytical variability. Creatinine output has
been reported to be fairly constant with time (variations within 10%) and to correlate
with lean body mass,126 but there are also reports suggesting that the correction for
creatinine in a urine spot sample could be misleading. Alternatively, the excretion rate of
the marker may be determined in a 24 hour urine collection. However, these collections
are subject to inevitable inaccuracies due to collection errors. With most markers,
similar results are obtained from either 24 hours, 2-hours, or spot urine (FMV, SMV)
collections.

It is long known that bone turnover and thus bone markers show significant diurnal
variations, with highest values in the early morning hours and lowest values during the
afternoon and evening.127 Most studies report daily amplitudes of 15 – 30%, although the
most pronounced diurnal changes have been communicated for CTX (Figure 8).128–134 It
should be borne in mind that the slope of diurnal changes is steepest during the morning
hours, which is usually the time at which urine samples are collected. This is true for
both urinary and serum markers. Controlling the timing of sample collection is therefore
a “bare necessity” for all types of markers (see also further below).
Figure 8

Diurnal variation. A–C: Comparison of three immunoassays measuring type I collagen

degradation products in urine. A: Deoxypyridinoline (DPD), B: Aminoterminal
crosslinked telopeptide (NTX), C: Carboxyterminal octapeptide (CTX). All values are ...

In addition, the effects of diet and food intake need to be considered with certain
markers. For example, the ingestion of hydroxyproline-rich foods, such as meat or
gelatine will markedly affect measurements of OHP in urine. 135 It is therefore necessary
to instruct patients to be on a collagen-free diet for at least 24 hours before collecting
their urine for OHP measurements. In contrast, urinary and serum DPD, NTX and CTX
are unaffected by collagen ingestion. Unlike most other bone markers, serum CTX values
are influenced by food intake, and samples for this marker need to be taken in the fasting
state.

The effect of acute exercise immediately or shortly before phlebotomy for bone turnover
markers has been studied with some markers appearing to rise by as much as 30–40% of
their baseline value, while others seem to be unaffected by these activities.

Variation Between Laboratories

Markers of bone turnover are now offered by a great number of commercial laboratories
and in some countries are widely used by practising physicians. A recent trial amongst
laboratories in Europe showed marked variability of most commercialised test kits, with
inter-laboratory coefficients of variation up to 40%. Results obtained from identical
blood and urine samples using the same assay and the same method differed by up to
5.6-fold between laboratories.136 Therefore, results from different laboratories cannot be
readily compared to each other, even if the same method and sample has been used.
Immunoassays for bone turnover markers should be included into routine proficiency
testing programs.

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Biological Sources of Variability

Intra-individual (i.e. biological) sources of variability are much harder to control then
technical aspects of variability. Many biological factors cannot be modified at all (e.g age,
gender, ethnicity etc.), while others are hard to control in clinical practice. Nevertheless,
every effort should be made to account for these factors when interpreting the results of
bone marker measurements.

Effects of Age and Changes in Sex Hormone Levels on Markers of Bone Remodelling
Once somatic growth subsides, the serum and urinary concentrations of most bone
markers return to a level somewhat lower than that seen during normal puberty and
growth. This stabilisation usually occurs during the 3rd decade and in healthy men, levels
of practically all markers remain more or less unchanged until 70 years of age. After that,
a slight increase is usually seen in formation markers such as serum BAP or OC, and
most resorption markers.84,137–139

Menopause is associated with a substantial acceleration in bone turnover, which is

mirrored by a 50–100% increase in both bone formation and bone resorption markers
(Figure 9).137,139–148 In early postmenopausal women, this increase in bone turnover may be
attenuated by calcium supplementation.149

Figure 9

Age-related changes in (A) bone specific alkaline phosphatase and (B) urinary collagen
type I aminoterminal crosslinked telopeptide (NTX). Pre, premenopausal women; peri
MP 1, early perimenopausal women; peri MP 2, late peri-menopausal women; post
MP, ...

Long-term treatment of women with oestrogen has been shown to reduce resorption
markers such as DPD and NTx to premenopausal levels and to correct secondary
hyperparathyroidism.150,151 A prospective study covering the perimenopausal transition in
healthy women suggests that changes in bone turnover start during late pre-menopause
with a decrease in bone formation, which is later followed by a rise in bone
resorption.152 It is now widely accepted that the accelerated rate of bone loss seen after
the menopause is mainly due to an uncoupling in bone turnover and an increase in bone
resorption. Studies employing specific bone markers indicate that bone turnover
continues to be increased (and to be associated with bone loss) even during late
menopause.153 In some postmenopausal women, but particularly in the very elderly, this
increase in bone turnover is often, but not always, found to be due to vitamin D and/or
calcium deficiency and secondary hyperparathyroidism. 154–156

In men, the pattern of age-dependent change in bone markers is quite different from
that observed in women. Markers of bone formation and resorption are high in men aged
20 to 30 years which corresponds to the late phase of formation of peak bone mass.
Thereafter they decrease reaching their lowest levels between 50 and 60 years (Figure
10).157–160 While there is general agreement on the age-related changes of bone turnover in
adult men between 20 and 60 years of age, data on bone turnover rates in men over the
age of 60 years are largely discordant. Based on recent cross-sectional studies,
concentrations of bone formation markers remained unchanged, decreased, or increased
with age in men over 60 years.157,158,160–163 Most studies evaluating age-related changes in
bone resorption markers observed an increase in serum and urinary
indices.157,158,160,163,164 However, this was not confirmed in other population-based studies
which showed no change in resorption indices with age. 159,165 A careful analysis of the
published data provides clues that may help to explain some of these discrepancies.
Differences in studies investigating age-dependent changes in bone formation and
resorption may be related to diverse population characteristics and sample size, to the
use of marker assays with different specificities, to age-related changes in renal and
hepatic function, and lastly to the inclusion of men with osteoporosis.

Figure 10

Age-related changes of biochemical markers of bone formation and resorption in men.

(A) Serum osteocalcin, (B) Serum bone-specific alkaline phosphatase, and (C) 24-h
urinary excretion of deoxypyridinoline. Reproduced from, Szulc, P, Garnero, P,
Munoz,...

As some biochemical markers of bone turnover are cleared via by the kidney (i.e. OC,
collagen telopeptides), age-associated decrease in glomerular filtration rate may affect
urinary and, to a larger extent, serum marker concentrations. In the presence of
decreased renal function, urinary excretion of bone resorption markers expressed as 24
hour output may be falsely decreased. Conversely, levels of markers corrected by urinary
creatinine can be falsely increased because of decreased creatinine filtration and
decreased muscle mass.166–168

Both static and dynamic histomorphometric studies suggest that bone formation
decreases with age in healthy men. In contrast, osteoclast function remains largely
constant with age. Consequently, decreased bone formation seems to be the principal
factor in bone loss in men.168,169 In the cohort of healthy men studied by Clarke et al.,
specific markers of bone resorption (urinary DPD) did not change with age, confirming
the histomorphometric findings that osteoclast function is preserved with increasing age
in these men.170 These results are in contrast to some of the previously mentioned cross-
sectional studies which report an age-dependent increase in serum and urinary indices
of bone resorption.157–160,163,164,167–169

As mentioned before, differences in population characteristics may, at least in part,

explain these inconsistencies. Furthermore, men with idiopathic osteoporosis are
histomorphometrically characterised by increased bone resorption with an increase in
eroded surfaces up to 90% when compared with age-matched controls. 171 Hence, the
observed increase in biochemical markers of bone resorption in population-based
studies might also be due to the heterogeneity of men investigated including men with
osteoporosis.

Taken together it remains controversial to what extent biochemical markers of bone

resorption change as a function of age in men over 60 years. Observational studies
investigating age-dependent changes in bone marker levels indicate that in elderly men
there seems to be an imbalance in bone turnover with increased bone resorption and
stable bone formation after the age of 60. As biochemical markers have been shown to be
negatively correlated with BMD, this imbalance in bone turnover may, at least in part, be
responsible for the age-related bone loss in men. 158,167 However, confounding factors such
as population characteristics, specificity of bone marker, as well as estimates of renal and
liver function have to be taken into account when evaluating age-dependent changes in
bone turnover.

Effects of Diurnal Variation on Bone Marker Measurements

Diurnal variation appears not to be affected by age, menopause, physical activity or

season. Although in postmenopausal women bone turnover is higher than in
premenopausal women, the circadian variation is similar for both pre- and
postmenopausal women and, thus, is not thought to be influenced by sex
hormones.138,172,173 The aetiology of diurnal variation is unknown. Several hormones, such
as parathyroid hormone, growth hormone, or cortisol show diurnal changes and may
therefore be involved in the generation of diurnal changes in bone
metabolism.175 Independent of this, there is wide agreement that controlling the time of
sampling is crucial in order to obtain clinically relevant information from bone markers.
Most biochemical markers show significant diurnal variations, with highest values in the
early morning hours and lowest values during the afternoon and at night. This has been
well documented for most urinary markers and amplitudes usually vary between 20 and
30 % (Figure 8).127 Serum markers usually show less pronounced changes during the day
than urine based indices. However, discrepant results have been reported for serum
CTX. Wichers et al. have reported daily amplitudes of serum CTX of up to 66%, while
others describe smaller changes (Figure 8D).134

Figure 8D

Diurnal variation in serum CTX levels in six healthy male volunteers. On average, the
peak value was 66% higher and the nadir was 60% lower than the calculated daily mean.
Reproduced from, Wichers M, Schmidt E, Bidlingmaier F, Klingmüller ...

Effect of Low-Frequency Biological Rhythms on Bone Marker Measurements

Intra-individually, biochemical markers of bone turnover not only vary within a single
day but in most cases also between consecutive days. This phenomenon is called
between-day or day-to-day variability and is apparently due to genuine variations in
marker levels and not to analytical imprecision. In general, serum markers show less
day-today variability than markers of bone turnover measured in urine. 175 The day-to-day
variation in the urinary excretion of PYD and DPD, measured by HPLC and corrected for
creatinine, ranges between 16–26%.176 Similar results have been reported for free
pyridinoline by EIA (7–25%), for the N-terminal collagen type I telopeptide (NTX, 13–
35%), for the C-terminal collagen type I telopeptide (CTX, 12–35%) and for tartrate-
resistant acid phosphatase (TRAP, 10–12%). 175,177 Day-to-day variability adds considerably
to the total variation of biochemical markers of bone turnover and unlike diurnal
variation, cannot be controlled.

Bone turnover varies with the menstrual cycle with an overall amplitude of
approximately 10–20%.178,179 There is evidence to support the suggestion that bone
formation is higher during the luteal than the follicular phase, whereas bone resorption
is higher during the mid-follicular, late-follicular and early luteal phase. 179,181 Cyclical
changes in bone turnover have also been reported in postmenopausal women treated
with sequential oestrogen/gestagen regimens, showing decreases during oestrogen
treatment and increases during gestagen treatment. 182 In premenopausal women with
metabolic bone disease, menstrual variability should be taken into account, and the
timing for sampling is probably best during the first 3–7 days of the menstrual cycle.

Bone turnover and its regulation seem to vary with seasonal changes. Some studies have
shown that serum 25-OH vitamin D and urinary calcium are higher in summer than in
winter, and that parathyroid hormone levels show inverse changes. 182,183 More recently,
seasonal changes were also described for markers of bone metabolism, with a 20–30 %
lower turnover rate in summer than in winter (Figure 11)184 The increase in bone
turnover in winter may be due, at least in part, to subclinical vitamin D deficiency.

Figure 11

Seasonal variability of serum bone specific alkaline phosphatase (S-BAP) and urinary
deoxypyridinoline (U-DPD). Values are presented as the percent change from the annual
mean (±SEM). The number and width of representative intervals of each bone ...

Effects of Somatic Growth

During early childhood and then again during the pubertal growth spurt, biochemical
markers of bone turnover are significantly higher than during adulthood. 188 In girls, peak
bone marker levels are observed approximately two years earlier than in boys, and
oestradiol seems to be the major determinant of the increase in bone turnover. In men
between 20 and 30 years of age, bone turnover markers are usually higher than in
women of the same age bracket. After the age of 50, most bone turnover markers tend to
increase with further ageing, but less in men than in women. In the latter, the age-
related increase in bone turnover is more pronounced due to the menopause, when both
bone resorption and formation increase by about 50–100 %. 186,187

Other Biological Sources of Variability

A number of non-skeletal diseases have been shown to strongly affect bone turnover
markers. These conditions mostly relate to impairments in the clearance and/or
metabolism of the components measured. Thus, even moderate impairment of renal
function (GFR 50 ml/min) has been shown to have significant effects on the serum levels
of OC, of BSP, and of the collagen type I telopeptides (NTX, CTX). 108,188,189

In summary, numerous factors influence bone turnover, but there are even more sources
of variability that need to be taken into account when measuring biochemical markers of
bone turnover. To minimise some of the limitations linked to pre-analytical and
analytical variability, standardised sampling and sample handling are mandatory to
obtain reliable results. Controllable factors such as the mode of sample collection,
sample handling and storage, diurnal and menstrual rhythms, pre-sampling exercise and
pre-sampling diet should be taken care of wherever possible. Laboratories are
encouraged to establish their own reference ranges and to use gender and age specific
reference intervals. In order to further reduce variability, standardisation of bone marker
assays and routine proficiency testing programs are strongly recommended.

Go to:

How to Deal with Variability

If bone markers vary so much, how can we ever use them in our patients? Admittedly,
most data on the use of bone markers have been generated in large populations or
controlled clinical trials, which may not be representative of a routine clinical setting.
However, bone markers can well be used in individual patients if methods are used that
account for potential sources of variability and thus integrate the inherent limitations of
bone markers measurements.

The Concept of Least Significant Change (LSC)

Numerous biological factors affect bone turnover and therefore bone marker levels
(Table 2). As a rule, markers showing large changes in response to disease processes or
interventions also show substantial degrees of biological variability. In the clinical
setting, variability of bone markers should be of particular concern when it comes to
serial measurements, for example during therapeutic monitoring. Often, a moderate
reduction in a bone resorption marker is believed to be the effect of anti-resorptive
treatment, when it really should be attributed to non-specific variability. However, a true
(“significant”) response in either BMD or bone turnover can only be assumed, when
within a single individual the change in signal is greater than the imprecision of the
measurement. This change is called the “LSC”. LSC can be defined for various levels of
confidence (e.g. 80% or 95%) and in large depends on the short- and long-term within
subject variability (CV) of a given marker. The CV of bone formation markers is lower
than that of most bone resorption markers, and so is their LSC. Thus, for formation
markers, a change >25% should under regular circumstances be considered significant,
while for most bone resorption markers (serum and urine) LSC is around 60–80%.

Another phenomenon to be considered when interpreting any serial measurement is the

regression to the mean. This effect is independent from biological variability and relates
to the changes in extreme baseline values.

The pronounced variability and heterogeneity of bone markers makes it difficult to

determine precise thresholds or cut-offs for practical use in individual patients. In
clinical medicine, two interrelated approaches are often used to assess the clinical
significance of a change. These include a comparison of the actual difference to a
predefined cut-off, and a comparison of the measured value to a predefined range.

The first method defines certain cut-off levels that a change in a marker must exceed to
be considered “clinically significant”. Some models apply LSC or similar statistical
approaches to define a-priori cut-offs.190 Other models have used the placebo and
treatment groups of large randomised controlled trials (RCTs) and calculated the
fracture incidence according to the change in a marker above or below a certain cut-off.
For example, using data from the fracture intervention trial, Bauer et al. demonstrated
that alendronate-treated women with a reduction in serum BALP of 30% or more at 1
year had fewer hip and non-spine (but not vertebral) fractures than alendronate-treated
women with a 1 year change in serum BALP of less than 30%. However, when compared
to the placebo group, there was no difference in the incidence of vertebral fractures
between the alendronate-treated groups (>30% vs. <30% change at 1 year). In this
instance, therefore, setting a 30% cut-off helped to define – a posteriori– a valid
response to alendronate treatment for non-spine and hip fractures, but appears to be
useless for vertebral fractures.194

Another widely used method to assess therapy-induced changes in laboratory

parameters is to compare the actual marker level to a predefined range (similar to the T
and Z score approach in bone mineral density [BMD]). In most instances, this range will
be the ‘reference’ or ‘normal’ range, which may or may not be standardised between
methods and/or laboratories. In the bone field, most people would agree that patients
with accelerated bone turnover are likely to benefit from anti-resorptive treatment if
their bone markers return to the respective ‘normal’, i.e. premenopausal range. Such a
‘normalisation” would be considered a valid response, while changes that do not lead to a
return of the marker into the reference range would be labelled as an invalid response.
Furthermore, a reduction of the marker below the reference range could be indicative of
over-treatment.

A recent paper by Sambrook et al. illustrates the usefulness of this approach. In a double
masked and double placebo controlled study, the authors compared the effects of
alendronate 70 mg once weekly with raloxifene 60mg daily on markers of bone turnover
in 487 postmenopausal women with low BMD. 193 Both anti-resorptive agents reduced
serum OC and urinary NTX-I levels after 1 year of treatment, but the effect was more
pronounced for alendronate. Large RCTs have shown that both alendronate and
raloxifene reduce fracture risk in women with low BMD. It is therefore interesting to
note that in the head-to-head trial by Sambrook et al., both alendronate and raloxifene
returned bone markers into their respective reference ranges. 192

The problem with this method is that reference ranges for most markers have not been
well defined. Also, a reduction into the ‘normal’ range can only be achieved if the pre-
treatment values are abnormally high, which is the case in less then 50% of patients with
osteoporosis. Clearly, the approach is also invalid for anabolic treatments, which
increase both bone formation and resorption. Finally, standardisation of most assays is
presently insufficient to provide a basis for a wider application of such a reference-based
approach.136

Go to:

Footnotes
Competing interests: None declared

Go to:

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