VIEWPOINT

Salivary biomarkers in cardiovascular disease: An insight

into the current evidence
Eshak I. Bahbah1,2, Christa Noehammer1, Walter Pulverer1, Martin Jung1 and
Andreas Weinhaeusel1
1 AIT Molecular Diagnostics, Center for Health & Bioresources, AIT Austrian Institute of Technology GmbH, Vienna, Austria
2 Faculty of Medicine, Al-Azhar University, Damietta, Egypt

Keywords
cardiovascular disease; CK-MB; C-reactive
protein; salivary biomarkers; troponin
Correspondence
A. Weinhaeusel, AIT Austrian Institute of
Technology GmbH, Giefinggasse 4, 1210
Vienna, Austria
Tel: +43 50550-4455
E-mail: andreas.weinhaeusel@ait.ac.at
(Received 19 August 2020, revised 28
November 2020, accepted 23 December
2020)
doi:10.1111/febs.15689
Cardiovascular diseases (CVDs) are the most common cause of mortality
worldwide. In acute cardiovascular conditions, time is a crucial player in
the outcomes of disease management. Given the ease and noninvasiveness
of obtaining saliva, salivary biomarkers may provide a rapid and efficient
diagnosis of CVD. Here, we reviewed the published data on the value of
salivary molecules for diagnosis of CVD, especially in acute care settings.
In this review, we show that some biomarkers such as salivary creatinine
kinase myocardial band, C-reactive protein, troponin-1, and myoglobin
exhibited promising diagnostic values that were comparable to their serum
counterparts. Other molecules were also investigated and showed contro-
versial results, including myeloperoxidase, brain natriuretic peptide, and
some oxidative stress markers. Based on our review, we concluded that the
clinical use of salivary biomarkers to diagnose CVD is promising; however,
it is still in the early stage of development. Further studies are needed to
validate these findings, determine cutoff values for diagnosis, and compare
them to other established biomarkers currently in clinical use.

Introduction
Cardiovascular diseases (CVDs) are the most common
cause of mortality; both globally and in the United
States. Solely, they were responsible for 20% and 24%
of the total age-matched worldwide liability of disease
in 2016, respectively. Among CVDs, the most common
is ischemic heart disease [174 million disability-ad-
justed life-years [DALYs; crude range 170–180 mil-
lion]), then stroke [116 million DALYs (crude range
111–121 million)] [1]. In the USA, 116.4 million, or
46% of adults, are estimated to have hypertension,
with ~ 2303 deaths owing to CVD daily and 389.4
daily deaths related to stroke alone. In Europe,
4 475 990 individuals were estimated to die from CVD
in 2017 [2]. By 2030, the excepted direct medical costs
for CVD will reach $818 billion in comparison with
$273 billion in 2010 [3].
Acute myocardial infarction (AMI) is a common
cardiovascular emergency with a high case-fatality
ratio. The cornerstone for emergency diagnosis of
AMI is electrocardiography (ECG) in addition to anal-
ysis of the classic cardiac biomarkers, including tro-
ponin I (TnI), serum creatine kinase myocardial band

Abbreviations
8-OHdG, hydroxydeoxyguanosine; AHSG, alpha-2-HS-glycoprotein; AMI, acute myocardial infarction; ASA, alcohol septal ablation; BNP, B-
type natriuretic peptide; CAT, catalase; CHD, chronic heart disease; CK-MB, Creatinine kinase myocardial band; CPK, Creatine
phosphokinase; CRP, C-reactive protein; CVD, cardiovascular disease; DCFH-DA, 2’7’ dichlorodihydrofluorescein diacetate; HDL, high-density
lipoprotein; IMA, ischemic modified albumin; LDL, low-density lipoprotein; LR, logistic regression; MDA, malondialdehyde; MMP, matrix
metalloproteinase; MPO, myeloperoxidase; MYO, myoglobin; NES, neuron-specific enolase; TG, triglyceride; TIMPs, tissue inhibitors of
metalloproteinases; TnI, troponin I; VLDL, very-low-density lipoprotein.

The FEBS Journal (2021) ª 2020 Federation of European Biochemical Societies 1

Salivary biomarkers in cardiovascular disease
(CK-MB), total CK, and myoglobin (MYO) serum
concentrations [4]. The time factor plays a crucial role
in the management plan of the patient. According to
Diercks et al. [5], every minute of delay in treating
AMI augments the mortality rate. Given the swift,
easy, and noninvasive procure to obtain saliva, its
constitutes of organic and inorganic molecules
(derived from the local capillary blood), exfoliated
cells, and microbes made it a potential source for
diagnosis of AMI in the emergencies and assessment
of health [6].
Currently, it is not clear if saliva is a good and suit-
able substrate to enable diagnostic improvements.
Nevertheless, for sure, there is the advantage that sal-
iva is noninvasively accessible and can be collected
directly by patients who are not handicapped or physi-
cally constrained [7]. Additionally, saliva can be col-
lected with the support of, for example, family
members or nursing staff from youngest babies and
aged individuals in old people’s homes, respectively.
Therefore, saliva-based diagnostics offers many
options from lifestyle to all variants of professional
medical testing [8]. For the professional medical set-
ting, we found that ACS’s saliva-based diagnostics
might be a very interesting setting for application for
excluding myocardial infarction at the general practi-
tioner or in retirement homes [9,10]. When a first diag-
nostic result/decision is relevant, saliva might be an
attractive analyte with the above-mentioned technical
and practical advantages.
This review aimed to summarize current knowledge
and provide a basis for researchers in the applied diag-
nostics area to evaluate CVD and ACS as a potential
field of application of a noninvasive, for example, sal-
iva-based diagnostics. We see a high potential and
need to improve and to enable early diagnosis in those
settings. Especially, the use of simple, reliable assays
and markers, and complete solutions as given in ‘Point
of Care’ (PoC) Systems have the potential to support
decision making, for example, in-home care, old peo-
ple’s or nursing homes or at hospitals/general practi-
tioners lacking a laboratory infrastructure [11,12].
Even during transportation to clinics in ambulances
when professional personnel is available, saliva might
be an option to run a simple test. In addition, such
assays would offer simple surveillance of MI patients
like in postrehabilitation support at different clinical
sites like in convalescent homes, when nursing staff
might more frequently come into a situation to clarify
in the quickest possible manner if persons have, for
example, an MI or not. Not least, saliva-based assays
should be well suited for screening and risk stratifica-
tion or for monitoring therapy response/success [13].
2 The FEBS Journal (2021) ª 2020 Federation of European Biochemical Societies
E. I. Bahbah et al.
From the chemical point of view, saliva is less complex
compared to blood, which might be on one side a
potential advantage; on the other side, however, this
has the disadvantage that biomarkers of interest are
found on low concentrations [14]. Although nowadays
very sensitive analytical methods for different classes
of biomarkers are available, the presence and concen-
tration of markers in saliva samples is of utmost
importance for evaluation and deciding on further
developments. In this review, we have summarized
findings from literature focusing on known analytes
and markers for ACS diagnostic applications.
Materials and methods
In the current review, we aimed to retrieve all original arti-
cles that investigated the changes in salivary biomarkers in
patients with various CVDs. Following a preliminary
search to identify the common biomarkers, we synthesized
the following search strategy for PubMed search engine
from inception to December 2019, and then, it has been
updated in November 2020 (Saliva OR Salivary OR ‘Sali-
vary Biomarker’) AND (Cardiovascular OR CVD OR
‘Coronary Heart Disease’ OR CHD OR CAD OR
‘Myocardial Infarction’). We aimed for the search strategy
to be broad and sensitive to retrieve any relevant article on
the topic. The search results were imported to EndNo-
teTMX8 (Clarivate Analytics, London, UK) software for
screening. The full texts of potentially relevant articles were
retrieved for further inspection.
The eligible articles underwent further scrutiny to con-
firm eligibility for inclusion. Then, we extracted the follow-
ing information: first author, year, sample size, group
allocation, tested parameters, biochemical analysis meth-
ods, and main findings (including the P-value of signifi-
cance). The extracted data were summarized, and the
overall findings based on evidence synthesis from these
studies are summarized in Fig. 1.
Results
The overall database search retrieved 2532 unique cita-
tions. Following title and abstract screening, 83 full
texts were retrieved for further scrutiny. Finally, 22
articles were selected for final inclusion in the current
review. The majority of included studies focused on
ischemic heart disease, while fewer studies included
patients with hypertrophic cardiomyopathy, hyperten-
sion, and heart failure. The criteria of the evaluated
biomarkers are illustrated in Table 1. The characteris-
tics and main findings of the included studies are sum-
marized in Table 2. Figure 2 summarizes the role of
these markers in the process of atherosclerosis and
myocardial infarction.
E. I. Bahbah et al.
Fig. 1. Summary of the results of reviewed studies about the
salivary levels of the most promising biomarkers for CVDs. Green
color:Parameters with agreement among the reviewed studies
over their direction of change in CVDs patients. Pale color:
Parameters with contradictory findings among the reviewed
studies over their direction of change in CVDs patients.
Creatinine kinase myocardial band (CK-MB)
CK-MB is an isoenzyme for creatine kinase that cat-
alyzes the transphosphorylation of phosphate from
creatine phosphate to ADP. It inhabits the cytosol
and enables the movement of high-energy phosphates
back and forth from the mitochondria. CK-MB is
cardiac-specific owing to its primary presence in the
myocardium, although small amounts are released
from skeletal muscles. Its serum levels upsurge follow-
ing the onset of chest pain by 6–12 h, summit within
16–24 h, and are restored to normal levels (5–
25 IU
L1) within 24–48 h. Because of its brief half-
life, it is helpful for evaluating re-infarction. The
degree of elevation commonly reflects the extent of
the infarction. However, this investigation demands
time that could defer treatment and affect the out-
come.
The FEBS Journal (2021) ª 2020 Federation of European Biochemical Societies
Salivary biomarkers in cardiovascular disease
The presence of CK in saliva could confer a quick,
less invasive investigation and lower cost. Mirzaii-Diz-
gah et al. assayed the serum and unstimulated whole
saliva of AMI patients (N = 30) to measure the level
of creatine phosphokinase (CPK) and CK-MB in com-
parison with healthy participants (N = 30). They ana-
lyzed CPK and CK-MB using the International
Federation of Clinical Chemistry method and
immunoinhibition assay, respectively. The results
showed that both serum and salivary levels of CPK
and CK-MB were higher (P < 0.01) in the AMI group
compared to healthy controls. There was also a signifi-
cant positive correlation between serum and salivary
levels of CPK and CK-MB [15,16]. Foley et al.
reached similar conclusions. They performed
immunosorbent assays of salivary samples from 21
patients before alcohol septal ablation (ASA) for the
management of hypertrophic cardiomyopathy. They
found that CK-MB was increased 70-fold at 8–16 h.
The mean salivary CK-MB concentration floated
uphill early (P > 0.05) and was restored to baseline
levels afterward [17]. Using Luminex bead array multi-
plex testing and lab-on-a-chip methods, the screening
capacity of a biomarker panel containing CK-MB fur-
ther surpassed the ECG alone [12]. However, serum
biomarkers exhibited higher AMI sensitivity and speci-
ficity compared to saliva, as concluded by logistic
regression (LR) analysis in the study by Miller et al.
[18] on 92 patients within 48 h of the start of cardiac
symptoms in comparison with 105 controls. Ebersole
et al. [19] revealed that measuring serum CK-MB is an
excellent test for AMI patients regardless of oral
health; however, its salivary component cannot be con-
sidered a differentiator.
C-reactive protein (CRP)
CRP is an acute inflammatory protein that is mainly
synthesized in hepatocytes. It surges up to 1000-fold at
sites of infection or inflammation [20]. Recent evidence
emphasizes inflammation as a strategic pathogenic
mechanism in the evolution of atherosclerosis and
increasing the risk for atherothrombotic CVD
events. CRP is implicated in promoting vascular
inflammation, endothelial injury, and heart diseases
[21]. The aforementioned study by Foley et al.
recorded significant elevations (P < 0.02) of salivary
CRP (up to 2.0- or 3.5-fold), as well as serum CRP
(P < 0.001). Similarly, they noted significant associa-
tions (r = 0.8) between serum and salivary CRP levels
[17]. Labat et al. sampled the saliva and plasma of 259
individuals and performed a carotid ultrasound, assess-
ment of pulse wave velocity, and blood pressure
3
Salivary biomarkers in cardiovascular disease E. I. Bahbah et al.

Table 1. Biochemical criteria of salivary biomarkers, investigated in CVDs.

Gene Molecular
location mass UniProt Data Biochemical functions

CK-MB 14q32 87 kDa Entry: I6LWZ5 CK-MB catalyzes the transphosphorylation of phosphate from creatine phosphate
Gene: CK-MB to ADP. It enables the movement of high-energy phosphates back and forth
Length: 54 from the mitochondria
amino acids
CRP 1q23.2 25 106 Da Entry: P02741 CRP recognizes foreign pathogens and damaged cells of the host and to initiate
Gene: PTX1 their elimination by interacting with humoral and cellular systems in the blood
Length: 224
amino acids
TnI 19q13.4 24 kDa Entry: P19429 TnI binds to actin in thin myofilaments to hold the actin-tropomyosin complex in
Gene: TNNI3 place
and TNNC1
Length: 210
amino acids
MPO 17q22- 146 kDa Entry: P05164 MPO exists in the neutrophil azurophilic granules and the lysosomes of
24 Gene: MPO monocytes, which plays a role in killing some bacteria
Length: 745
amino acids
MYO 22q13.1 16.7 kDa Entry: P02144 MYO exists in muscles and transports oxygen from red blood cells to the
Gene: MB mitochondria of muscle cells
Length: 154
amino acids
Brain 1p36.2 12 kDa Entry: P16860 BNP promotes diuresis, natriuresis, vasodilation of the systemic and pulmonary
natriuretic Gene: NPPB vasculature
peptide Length: 134
amino acids
MMP-8 11q22.2 53 kDa Entry: P22498 MMP-8 is involved in the breakdown of extracellular matrix in during embryonic
Gene: MMP8 development, reproduction, and tissue remodeling
and CLG1
Length: 467
amino acids
Adiponectin 3q27.3 27 kDa Entry: Q15848 Adiponectin protects the cardiovascular system during stress and has an anti-
Gene: inflammatory role
ADIPOQ
Length: 247
amino acids
Irisin 1p35.1 12 kDa Entry: Irisin participates in the development of brown fat and regulating the functions of
Q8NAU1 skeletal muscle, heart, and liver cells
Gene: Fndc5
Length: 121
amino acids

measurement on every subject included in the study.
They noticed a significant positive correlation between
salivary CRP and mean pulse pressure, blood pressure,
and intima-media width. In an adjusted analysis for
age and sex, salivary CRP was correlated with carotid
plaques and serum CRP levels (r = 0.73, P < 0.0001)
[22].
Another group analyzed serum and saliva CRP in
92 AMI patients after 48 h of the onset of retroster-
nal pain and 105 healthy participants via immunoas-
say. LR identified salivary CRP as the most
predictive biomarker of AMI [18]. In concordance,
Floriano et al. [12] concluded that a salivary biomar-
ker panel containing CRP demonstrated high diag-
nostic capability (P < 0.0001), and when combined
with ECG, it had higher diagnostic capacity
(AUC = 0.96) for AMI than ECG alone. Moreover,
Al-Rawi et al. [23] found that 20 (50%) participants
with chronic heart diseases (CHD) had positive sali-
vary CRP, while only three (7.5%) healthy controls
had positive CRP (OR = 1.6, 95% CI: 0.6–4.25;
P < 0.01). Ebersole et al. [19] revealed that regardless

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E. I. Bahbah et al. Salivary biomarkers in cardiovascular disease

Table 2. Summary of the results of the reviewed studies.

Study ID Subjects Tested parameters Analysis method Findings

Al-Rawi and 40 ischemic heart Cholesterol profile, Spectrophotometry, Significant differences between the two groups in
Shahid disease and 40 MDA, UA, SOD, colorimetric assay, terms of salivary total cholesterol (P = 0.003),
2017 controls LDH, and CRP agglutination assay serum HDL (P = 0.03), salivary HDL (P = 0.001),
serum MDA (P < 0.001) and serum LDL
(P < 0.001). Other factors (salivary SOD, UA,
LDH, and CRP) were comparable between the
two groups
Ebersole 92 AMI and 111 CRP and Immunoassay Salivary adiponectin was increased by 50% in the
et al. 2017 controls adiponectin AMI group; however, this was statistically
insignificant (P = 0.58). While CRP levels were
significantly raised in both serum and saliva
(P < 0.0001)
Nguyen 16 ACS and 16 MDA Spectrophotometry Salivary MDA was significantly higher in the ACS
et al. 2016 controls group than in the control group (1.69  0.48 vs.
0.36  0.44; P < 0.001)
Nguyen 24 ACS and 24 8-OHdG, PC, MDA, ELISA Salivary 8-OHdG (> 8 folds), MDA (17 folds), PC
et al. 2016 controls TAOC (1.5 folds), and TAOC (2.5 folds) levels were
significantly higher in ACS group than in healthy
controls (P < 0.001).
Zhang et al. 63 HF and 51 controls Galectin-3 ELISA and The galectin-3 concentrations were significantly
2016 immunoassay elevated in saliva (2.8 to 2510 ng
mL1) and
serum (3.3 to 121.5 ng
mL1) samples of patients
with HF compared with controls (P < 0.001 and
P < 0.0001, respectively)
Khalighi 34 coronary artery Homocysteine HPLC Salivary homocysteine levels were
et al. 2015 disease and 32 0.24  0.056 mM in CAD patients and
controls 0.023  0.013 mM in the control group with a
statistically significant difference (P < 0.001)
Rathnayake 200 AMI and 200 MMP-8 and MPO ELISA, The mean salivary levels of MMP-8 (543 vs.
et al. 2015 controls immunofluorescence 440 ng
mL1, P = 0.003) and MPO (1899 vs.
assay 1637 ng
mL1, P = 0.02) were higher in control
subjects compared to MI patients
Aydin et al. 11 AMI and 14 controls Irisin ELISA The salivary irisin level increased early (1 h:
2014 302.64  60.56 ng
mL1; 12 h:
264.46  20.56 ng
mL1) and started 9
decreasing gradually after 12 h, 18 h:
250.68  13.22 ng
mL1; 48 h:
238.17  6.51 ng
mL1) in AMI patients and
were significantly different from controls
(P < 0.05)
Ebersole 92 AMI and 111 TnI, CK-MB, and Immunoassay The salivary CRP (P < 0.004), TnI (P < 0.01), and
et al. 2014 controls CRP CK-MB (P < 0.01) levels were significantly higher
in AMI patients compared to controls, during the
48 h interval postcardiac event
Miller et al. 92 AMI and 102 CRP, sICAM, and Immunoassay The salivary levels of adiponectin (OR = 1.68, 95%
2014 controls adiponectin CI: 1.17, 2.40; P = 0.004), sICAM (OR = 1.92,
95% CI: 1.26, 2.93; P = 0.002), and CRP
(OR = 1.68, 95% CI: 1.33, 2.13; P < 0.0001) were
significantly higher in AMI patients, compared to
controls
Zheng et al. 18 various CVD and 20 AHSG western blotting The AHSG levels were significantly lower
2014 OSA controls (P < 0.01) in the CVD group than the non-CVD
group
Labat et al. 81 hypertensive and RP, PGE2, LTB4, ELISA and The salivary levels of MMP-9 (P = 0.002), PGE2
2013 178 controls MMP-9, spectrophotometry (P = 0.04), and CRP (P = 0.002) were significantly

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Salivary biomarkers in cardiovascular disease E. I. Bahbah et al.

Table 2. (Continued).

Study ID Subjects Tested parameters Analysis method Findings

creatinine, different between hypertensive and control

lysozyme levels subjects. While the levels of LTB4 (P = 0.09) and
creatinine (P = 0.14) were comparable between
the two groups
Mirzaii- 30 AMI and 28 controls TnI ELISA At 12 h of MI onset, the resting saliva
Dizgah and concentrations of cTnI (median: 0.40, IQR: 0.17–
Riahi 2013 0.62), but not stimulated saliva cTnI were
significantly higher than in controls (median 0.19
(0.08–0.27 ng
L1); P < 0.01
Rubio et al. 10 AMI and 10 controls CAT level Spectrophotometry There was no difference in salivary CAT activity
2013 between controls and AMI subjects at 24 h or at
48 h postdiagnosis (P = 0.15)
Toker et al. 60 AMI and 40 controls Ischemia modified Manual colorimetric Salivary IMA levels were significantly higher in the
2013 albumin (IMA) assay first (1.30  0.1 vs. 0.92  0.1) day of AMI
levels patients compared to controls (P < 0.001)
Foley et al. 19 hypertrophic TnI, CK-MB, BNP, Immunoassay TnI, CK-MB, MYO, and CRP rose in serum 2- to
2012 cardiomyopathy and MYO, MMP-9, 812-fold after ASA (P < 0.01). Significant
97 controls CRP, MPO elevations of 2.0- to 3.5-fold were observed with
CRP and TnI in saliva (P < 0.02).
Mirzaii- 32 AMI and 32 controls CK-MB Immunoassay CK-MB levels showed a significant elevation in
Dizgah saliva and serum of patients with acute MI
et al. 2012 compared to healthy controls
Shen et al. 85 AMI and 388 a-amylase Quantitative enzyme Initial salivary a-amylase activity in the AMI group
2012 controls kinetic method (266  127.6 U
mL1) was significantly higher
than in the control group (130  92.8 U
mL1,
P < 0.001)
Budnelli 47 AMI and 17 controls Activated MMP-8 Immunoassay Salivary MMP-8 was higher in non-AMI than in
et al. 2011 AMI patients, but a significantly higher percentage
of AMI patients had activated neutrophil MMP-8
(P < 0.001)
Mirzaii- 30 AMI and 30 controls CK-MB Spectrophotometry CK-MB levels showed a significant elevation in
Dizgah and saliva (72.41  14.15 vs. 8.16  0.54 U
L1;
Jafari-Sabet P = 0.004) and serum of patients with acute MI
2011 compared to healthy controls
Al-Rawi and 50 ischemic stroke, 25 NSE ELISA Salivary NSE concentrations showed higher NSE
Atiyah 2009 control, 25 DM, 25 values in patients with ischemic stroke compared
IHD, 25 hypertension to healthy controls (4.5  1.6 vs.
2.9  0.5 mg
L1)
Floriano 41 AMI and 43 controls CRP, MYO, and ELISA and The saliva-based biomarker panel of CRP, MYO,
et al. 2009 MPO immunoassay and MPO exhibited significant diagnostic
capability (AUC = 0.85, P < 0.0001) and, in
conjunction with ECG, yielded strong screening
capacity for AMI (AUC = 0.96)

the oral health, salivary CRP exhibited good sensitiv-
ity in identifying AMI patients.
Troponin I (TnI)
TnI (or the troponin complex) is a protein complex
that participates in muscle contraction. It exists at reg-
ular intervals within thin myofilaments of striated mus-
cles (skeletal and cardiac) and is bound to the
protein tropomyosin. Individual protein performs
diverse purposes: Troponin C attaches to calcium ions
to yield a conformational change in TnI. Troponin T
attaches to tropomyosin, interconnecting them to pro-
duce a troponin-tropomyosin compound, while TnI
attaches to thin actin filaments to grasp the troponin-
tropomyosin compound in place. Given the difference
between troponin T and TnI forms in the skeletal and
heart muscles, troponin can be used as a cardiac-speci-
fic biomarker. Cardiac TnI rises in the serum after 4 h
(peaks at 24 h) of chest pain and stays elevated for 7–

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E. I. Bahbah et al. Salivary biomarkers in cardiovascular disease

Fig. 2. Role of cardiovascular biomarkers in the process of atherosclerosis. PON1; Paraoxonase 1, APOA1; Apolipoprotein A1, IL;
Interleukins, MCP-1; Monocyte Chemoattractant Protein-1. HOCl; Hypochlorous acid, eNOS; Endothelial NOS, VCAM-1; Vascular cell
adhesion protein 1, ICAM-1; Intercellular Adhesion Molecule 1, ABCA1; ATP-binding cassette transporter.

10 days. It is considered one of the most specific pro-
tein biomarkers [24].
Mirzaii-Dizgah et al. assayed TnI in the saliva and
serum of 30 patients with AMI (at an interval of 0.5
and 1 day of the onset of AMI) and 28 healthy con-
trols. They found that in patients with AMI, the serum
and unstimulated salivary levels of TnI at both half-
day and day of the onset of AMI were significantly
higher in the AMI group, compared to controls.
Moreover, a significant correlation (r = 0.45,
P = 0.004) existed between resting saliva and serum
TnI levels [9]. In the aforementioned study by Foley
et al. on patients undergoing ASA for hypertrophic
cardiomyopathy, the authors found that serum TnI
increased at 16–24 h over an 800-fold increase
(P < 0.001). Moreover, significant increases of 2- to
3.5-fold were spotted with TnI in saliva (P < 0.02).
Similarly, Floriano et al. elucidated that the panel of
biomarkers contains TnI further exceeded the validity
of diagnosis AMI with ECG alone. Saliva-based panel
tests may complement ECG to offer a far more handy
and quick screening technique for diagnosing AMI
patients. Still, Ebersole et al. [19] disclosed that serum
TnI could be deliberated as an excellent assessment for
AMI patients regardless of oral health; however, its
salivary component cannot be considered for discrimi-
nation.
Myeloperoxidase (MPO)
MPO is a peroxidase enzyme produced by white blood
cells. It catalyzes the burst reaction in neutrophils and
the creation of reactive oxygen species. It plays a role
in activating latent forms of proMMP-8 and

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Salivary biomarkers in cardiovascular disease
proMMP-9 and suppressing tissue inhibitors of metal-
loproteinases (TIMPs) [25]. It participates in tissue
injury through a redox reaction, which is a component
of Virchow’s triad of thrombus formation. Thus, MPO
is considered proatherogenic and contributes to the
pathogenesis of CVD [26].
Miller et al. [18] found that serum and salivary
MPO levels were increased significantly in samples col-
lected 48 h following the onset of substernal ache
(P < 0.001, P = 0.013, respectively). Similarly, Folari-
ano et al. found that in unstimulated whole saliva, the
MPO median diseased/median control ratio was signif-
icantly higher (1.9 mM, P = 0.0008). Foley et al. [17]
revealed strong correlation coefficients for salivary
MPO (r range = 0.68–0.90 for the entire interval
points; P < 0.05). Nevertheless, these results were not
in line with Rathnayake et al. [27], who attributed ele-
vated salivary MPO to periodontal disease.
Myoglobin (MYO)
MYO is a cytoplasmic iron- and oxygen-binding
hemoprotein. It is found exclusively in cardiac muscles
and oxidative skeletal muscle fibers and encloses a
heme moiety that binds to oxygen reversibly. The
MYO gene expresses an individual polypeptide
sequence with one oxygen-binding site. It is considered
as an oxygen supply reserve in the musculature based
on the oxygen availability in myocytes. Moreover, it
functions as a buffer for reactive oxygen species and
hemostasis of nitric oxide [28].
Clinically, it is an early marker of AMI due to its
low molecular weight (16.7 kDa). MYO predictably
increases 2–4 h after chest pain onset, summits at 6–
12 h, and is restored to normal levels (0–85 ng
mL1)
within 24–36 h [29]. By examining the salivary and
serum samples of 43 AMI patients following 48 h of
retrosternal pain, Floriano et al. [12] found increased
levels of MYO in the serum and saliva of AMI
patients against healthy subjects (P < 0.05). Likewise,
Foley et al. revealed the rise of serum MYO consider-
ably postablation up to 11-fold after 8 h from symp-
toms start; nevertheless, the increase in salivary MYO
was less dramatic. Notably, a positive correlation
amongst serum and salivary MYO at 16 h existed
(r = 0.58, P = 0.04) [17]. However, Miller et al. [18]
found no significant difference in salivary MYO
between AMI and healthy control groups (P = 0.18).
Ebersole et al. [19] stated that although serum MYO
can be an accurate biomarker for AMI patients
regardless of oral health, however, its salivary con-
stituent cannot be considered for judgment.
8 The FEBS Journal (2021) ª 2020 Federation of European Biochemical Societies
E. I. Bahbah et al.
Brain natriuretic peptide (BNP)
BNP is a peptide hormone produced from ventricular
myocytes as a reaction to volume enlargement and the
augmented wall stress of the myocardium. It promotes
diuresis, natriuresis, and vasorelaxant effects, lessening
the level of endothelin in the circulation; and inhibits
the renin-angiotensin-aldosterone system and sympa-
thetic stimulation. BNP blood levels are raised in sev-
eral pathologic conditions, that is, myocardial fibrosis
and ischemia, ventricular remodeling, and overload
[30].
Examining 21 serum and salivary postalcohol septal
ablation patients’ samples, Foley et al. found that
baseline levels of salivary BNP were significantly infe-
rior to the mean control levels, then ascend after 16 h
to levels 2.3-fold at 48 h (P < 0.02). Conversely, the
mean serum BNP levels at the start point were signifi-
cantly above mean control values (P < 0.001) and des-
cended after 16 h [17]. Ebersole et al. concluded that
serum BNP could be used as an accurate detector for
AMI patients irrespective of oral health; however, its
salivary part is not reliable for discrimination [19].
Likewise, Miller et al. recorded significantly higher
serum BNP levels in the AMI group compared to
healthy controls (P < 0.001). Nevertheless, the salivary
BNP level was slightly higher among controls com-
pared with the AMI group, with a dim significant dif-
ference among both groups (P = 0.035) [18]. Floriano
et al. [12] found that the ratio between the medians of
the AMI group to the medians of controls in the
serum was 5.36. However, in saliva, it was only one
and without significant difference.
Oxidative stress biomarkers
Myocardial injury is associated with increased levels of
reactive oxygen species, that is, peroxide, superoxide,
and hydroxyl group [31,32]. Malondialdehyde (MDA)
is the product of lipid peroxidation of fatty acids and
might also be an outcome of the metabolism of arachi-
donate to prostaglandin H2 within different cell types.
The quantity of MDA within the tissues can be used
as a measure of lipid peroxidation. Cardiac myocytes
have an additional vulnerability to oxidative stress
compared to other cells due to their intrinsic proper-
ties. Hydroxydeoxyguanosine (8-OHdG) is an oxidized
form of deoxyguanosine, and its amount present
within a cell is an indicator of oxidative damage to
nucleic acids.
Al-Rawi et al. examined the serum and salivary
sample of 40 patients who had a recent acute coronary
syndrome event in comparison with 40 normal
E. I. Bahbah et al.
participants. They found that serum MDA concentra-
tion was significantly increased in 11 participants with
coronary heart disease vs. the control group
(P ≤ 0.01). Nguyen et al. found that salivary MDA
quantities were significantly elevated among patients
with both acute coronary syndrome and chronic peri-
odontitis than in those with only either one of them
than in healthy participants (P < 0.05). A positive cor-
relation existed between salivary MDA levels and peri-
odontal clinical parameters (P < 0.001). The same
authors found salivary 8-OHdG, MDA, and protein
carbonyl concentrations were significantly higher in
the patients with ischemic heart diseases and dental
diseases than in the healthy participants (P < 0.05).
Interestingly, the authors found a significant correla-
tion between salivary protein carbonyl concentrations
and salivary 8-OHdG concentrations (P < 0.05). Fur-
ther, a significant correlation also existed between sali-
vary MDA and all and other biomarkers for CVD
(P < 0.05) [33,34].
Other oxidative stress biomarkers, including 20 70
dichlorodihydrofluorescein diacetate (DCFH-DA),
were increased in the saliva of the AMI group vs.
healthy participants after 48 h of the onset of ret-
rosternal pain (P = 0.004). However, the change in
catalase enzyme (CAT) activity in the saliva between
both groups was absent at 24 and 48 h after the onset
of chest pain (P = 0.157). The correlation was nearly
significant between CAT48 and DCFH-DA48
(P = 0.053) [35].
Antioxidants like uric acid (UA) and superoxide
dismutase (SOD) play protective roles in the cardio-
vascular system due to their ability to neutralize reac-
tive oxygen species. UA is the final product of purine
metabolism that is chiefly metabolized by the xan-
thine-oxidoreductase enzyme. The level of serum UA
can change owing to dietary factors, drugs, or con-
genital or neoplastic diseases. SOD is an enzyme that
catalyzes the disproportionation of the superoxide
(O2) radical into hydrogen peroxide (H2O2). Al-Rawi
et al. [23] demonstrated that the mean values of serum
and salivary UA and SOD were slightly superior in
the CHD group than in healthy controls; however,
this difference was statistically insignificant (P > 0.05).
Metalloproteinases and Inflammatory cytokines
Matrix metalloproteinases (MMPs) are calcium-depen-
dent endopeptidases, which belong to the protease
enzyme family. There are ~ 23 different MMPs in the
human body. They degrade the extracellular matrix
proteins during organogenesis, tissue development, and
remodeling [36]. MMP-8, released from neutrophils, is
The FEBS Journal (2021) ª 2020 Federation of European Biochemical Societies
Salivary biomarkers in cardiovascular disease
involved in inflammatory conditions. They are also
overexpressed in macrophages, endothelium, and
smooth muscle cells and in atherosclerotic plaques. Its
collagenase action, hence alternative nomenclature col-
lagenase-2 or neutrophil collagenase, plays a role in
the conversion of stable plaques to unstable plaques
with consequent rupture [37].
Salivary MMP-8 levels are implicated in progressive
attachment loss in periodontitis [38]. Moreover, MMP-
9 increased in saliva during periodontal disease and
CVDs as well [39]. This could explain the underlying
poor oral health in AMI patients. Knocking down, the
expression MMP-9 in multiple animal models, was
prophylactic for CVD [40]. Buduneli et al. [41] found
that sera MMP-8, MMP-9, and TIMP-1, besides sali-
vary polymorphonuclear leukocyte type MMP-8, were
significantly elevated in the AMI group vs. healthy
group. In another study, correlating biomarkers and
sex, they found that MMP-8 was significantly elevated
in men compared to women regardless of myocardial
infarction status. MMP-8/TIMP-1 ratio was signifi-
cantly superior in men vs. women with myocardial
infarction [27].
Labat et al. found an association between MMP-9
and intima-media width between prostaglandin E2
(PGE2) and leukotriene B4 (LTB4) and decrease arte-
rial elasticity, and between lysozyme and increase
blood pressure [22]. These results were in line with
Floriano et al.’s findings; they found that in unstimu-
lated whole saliva, the ratio in median concentrations
of MMP-9 was the second utmost significant among
new biomarkers (P < 0.0029). Further, salivary soluble
intercellular cell adhesion molecule-1 (sICAM-1) was
significantly elevated in the AMI group compared to
healthy subjects. Conversely, salivary soluble CD40
ligand (sCD40L) and tumor necrosis factor a (TNFa)
were significantly lower in the AMI group vs. control.
The rest of the investigated inflammatory cytokines
including interleukins (IL-1b, IL-18, IL-6), monocyte
chemotactic protein 1 (MCP1), E-selectin, growth-reg-
ulated oncogene-alpha (Gro-a), and soluble vascular
cell adhesion molecule 1 (sVCAM-1), as well as their
salivary components, were nonsignificantly different
between the AMI and non-AMI groups [12].
Both Foley et al. and Miller et al. [17,18] revealed
significant correlations between the serum and salivary
levels of MMP-9. Miller et al. demonstrated a signifi-
cant increase in the level of MMP-9 in the AMI group
compared to healthy controls; however, the salivary
MMP-9 was not significantly different between both
groups. They also found no significant difference in
serum and salivary IL1B in both groups. Additionally,
TNF was significantly higher in the control group,
9
Salivary biomarkers in cardiovascular disease
besides the absence of a significant difference in serum
between both groups. Conversely, Rathnayake et al.
[27] concluded that MMP-8 and MMP-9 could not
distinguish between patients with or without AMI and
noted that oral conditions must be considered when
analyzing the levels of inflammatory salivary biomark-
ers.
Lipids and adipokines
Dyslipidemia is a major risk factor for atherosclerosis
and its complications, for example, angina pectoris
and myocardial infarction. The conventional method
to assess dyslipidemia is through measuring plasma
triglyceride (TG), total cholesterol, low-density
lipoprotein (LDL) cholesterol, very-low-density
lipoprotein (VLDL) cholesterol, and high-density
lipoprotein (HDL) cholesterol. Al-Rawi et al. studied
the periodontal health quality and the salivary lipid
profile in 40 ischemic heart disease patients vs. 40 non-
cardiac patients (controls). They found that the total
salivary cholesterol, TG, LDL, and VLD levels were
increased in ischemic heart disease patients vs. healthy
controls. However, salivary HDL was reduced in the
ischemic group, compared to controls [23].
Adiponectin is a peptide hormone released from the
adipose tissue and has an anti-inflammatory role.
Decreased adiponectin levels are prevalent in obesity-
related diseases, for example, type II diabetes, meta-
bolic syndrome, and CVD. Thus, it has become a
broadly accepted biomarker for these disorders since
its discovery in 1990 [42]. Of note, adiponectin protects
the cardiovascular system during stress and is consid-
ered a favorable therapeutic target [43]. Ebersole et al.
examined the saliva of patients within 48 h of an acute
AMI vs. control subjects. They found a significant
reduction of adiponectin levels in the serum of AMI
patients. Serum adiponectin in either group and sali-
vary level in the AMI group declined with rising body
mass index [44]. Floriano et al. [12] revealed that sali-
vary adiponectin was significantly higher, while serum
adiponectin was significantly lower among AMI
patients vs. control (P < 0.05). Table 2 summarizes the
results of the reviewed studies.
Others
Homocysteine is a sulfhydryl-containing amino acid
produced from methionine demethylation. Every rise
of 5 mM
L1 in homocysteine level raises the risk of
CHD incidents by about 20%, independently of other
CHD risk factors. The average value of plasma homo-
cysteine ranges from 7 to 14 mM in adults and is about
10
E. I. Bahbah et al.
6 mM in 3- to 14-year-old children [45]. Khaligi et al.
obtained blood and saliva samples from 34 partici-
pants with coronary artery diseases and 32 noncardiac
participants. The average plasma and salivary homo-
cysteine levels were significantly higher among coro-
nary heart disease patients vs. controls (P < 0.001).
Notably, a positive correlation between salivary and
plasma levels of homocysteine in either group was
found.
Other promising CVD biomarkers include galectin-
3, irisin, alpha-2-HS-glycoprotein (AHSG), ischemic
modified albumin (IMA), and neuron-specific enolase
(NSE). Zhang et al. tested the credibility of galectin-3
in the plasma and saliva as a biomarker for heart fail-
ure. The salivary and serum galactin-3 levels were sig-
nificantly higher (P < 0.001) in heart failure patients
compared to controls (Saliva: 282 vs. 601.3 and Serum:
7.8 vs. 16.5 ng
mL1 for normal controls and heart
failure patients, respectively). A positive correlation
between serum and salivary galectin-3 concentrations
was found [46]. Irisin is another biomarker produced
from cardiac myocytes and can be used as a biomarker
for AMI. The level of salivary and blood irisin begins
to decrease after 12 h from the onset of chest pain
then rises at 72 h. Aronis et al. [47] demonstrated that
there was no significant association between the level
of Irisin and the development of ACS; however, they
showed that increased irisin levels are associated with
the development of MACE in patients with established
coronary artery disease after PCI. On the other hand,
Abd El-Mottaleb et al. [48] proposed that irisin can be
used as a diagnostic marker for MI. Moreover, they
showed a negative correlation between irisin levels and
TNF-a, TnI, CK-MB, LDL-C, and TC. Interestingly,
it was observed that serum levels of irisin were signifi-
cantly negatively correlated with ST elevation and
QTc in rats at risk of MI [49]. Thus, it could be used
along with the classic cardiac biomarkers, including
TnI, CK-MB, and MYO. However, large-scale studies
are required to ensure the accuracy, percussion, sensi-
tivity, and specificity of this test for the diagnosis of
AMI [50]. In addition, Zheng et al. found that the
level of salivary AHSG was decreased among CVD
patients in comparison with healthy participants [51].
Likewise, the levels of the salivary biomarker (IMA
and NSE) were significantly higher in the AMI and
recent cerebrovascular accident groups compared with
normal participants [52,53] (Fig. 1).
In summary, Fig. 1 shows that the included study
had a consensus over the increased salivary levels of
CK-MB, CRP, MDA, and DCFA-DA in patients with
CVD. At the same time, they showed marked discrep-
ancies regarding the observed changes in the salivary
The FEBS Journal (2021) ª 2020 Federation of European Biochemical Societies
E. I. Bahbah et al.
levels of MMP-9, TnI, MPO, MYO, BNP, and Adi-
poQ. The latter outcomes need further investigation in
future studies to reach a consensus on their change
direction (increased or decreased) and also ascertain
their diagnostic value in CVD.
Discussion
Based on the aforementioned review of published data,
the clinical utility of salivary biomarkers to diagnose
CVD is promising but is still in the early stage of
development. Although relatively adequate data exist
on the utility of some salivary biomarkers as CRP,
CK-MB, MYO, and TnI, they still need further testing
on a large-scale to clearly elucidate their diagnostic
sensitivity and specificity. Other biomarkers still have
few data as MMP, oxidative stress, inflammation, and
adipokines and should be the axis of focus in addi-
tional studies. The most frequently successful biomar-
ker was CRP, and therefore, it may be suitable for
further clinical development.
However, we should note that there are several limi-
tations to the published evidence in this regard. First,
the reviewed studies used diverse designs and inclusion
criteria; therefore, comparative conclusions cannot be
acquired. Further, some studies had a relatively small
sample size (usually under 40 patients), which may
explain the lack of statistical significance in some out-
comes and the observed heterogeneity in the findings
of different studies. In addition, the used biochemical
analysis techniques to detect the same parameter var-
ied among the included studies, that is, immunoinhibi-
tion, ELISA, and other approaches were used in
diverse studies to assess the same outcome. The lack
of a standardized approach to measure salivary
biomarkers adds to the observed heterogeneity in the
above findings.
Recent guidelines of the European Society of Cardi-
ology [14] foresee the serial measurement of high sensi-
tivity cardiac Troponin (hs-cTn) in blood at 0 and 1
or 2 h after hospital admission of patients with acute
chest pain as a complement to clinical assessment and
ECG when it comes to diagnosis, risk stratification,
and treatment of patients with suspected ACS. There
is no doubt that blood-based protein biomarker testing
is the method of choice at this acute setting, as central
laboratory test can provide results in < 1 h and also
first blood-based hs-cTn I PoC Tests have recently
been shown to deliver comparable performance char-
acteristics to that of central laboratory hs-cTn I/T
assays [54,55]. Despite the above-mentioned clear
guideline for cTn testing, one has to be aware that
many cardiac pathologies other than MI also result in
The FEBS Journal (2021) ª 2020 Federation of European Biochemical Societies
Salivary biomarkers in cardiovascular disease
cardiomyocyte injury and, therefore, cardiac troponin
elevations. Along these lines, a multibiomarker
approach is certainly useful and the way to go in reli-
able differential diagnosis and risk stratification. As
specificity is an important aspect of CVD biomarkers,
the range of potentially useful biomarkers is here cer-
tainly not limited to proteins mentioned along with
this review but includes other promising biomarker
classes and types such as miRNAs, the microbiome,
and extracellular vesicles (EVs) as evident from numer-
ous studies [56–58]. Although saliva has its limitations
in the acute MI setting as it is a more distant body
fluid for detecting acute cardiac events and injury than
blood and acute patient’s salivary flow happens to be
impaired, saliva appears to be an attractive sample
matrix in the home care, general practitioner, or old
people’s home setting for regular risk stratification as
well as monitoring therapy response as it is readily
available and can be obtained noninvasively by non-
professionals. However, the saliva biomarker field is
still in its infancy and nowhere near as advanced as its
blood counterpart; there a more and more studies
coming up which demonstrate a good correlation
between blood and saliva biomarkers across various
biomarker classes and strongly support the concept of
noninvasive saliva-based diagnostics. As for blood
biomarkers, standardization in sample taking, as well
as sample pre-analytics, is, of course, also most crucial
for salivary biomarkers. Likewise, the stability of a
given salivary biomarker, as well as factors potentially
biasing the analysis of this marker, has to be thor-
oughly assessed when developing a diagnostic assay.
Currently, technical solutions for multiplexed analy-
sis in PoC devices or simple lateral flow devices are
under development. This will enable also multiplexed
analysis of samples; however, current knowledge based
on our review is sparse, and studies are needed to eval-
uate the potential for this use case as of (a) known
and established markers from blood—with all their
limitations, (b) a combination of CVD in the multi-
plexed analysis, and (c) novel biomarkers—for which
omics discovery strategies of different analytes in sal-
iva has to be done; to our knowledge—research is
missing or sparse; in addition, a combination of all
approaches (d) is also of interest; however, always
have in mind the technical feasibility of combining
analytes in PoC solutions and the practical applicabil-
ity, which is still quite different from the high-end
technical potential of a clinical or clinical research lab-
oratory.
Moving forward, we have some recommendations
for future studies. First, a standardized protocol
should be set to reduce the heterogeneity between
11
Salivary biomarkers in cardiovascular disease
studies in the field. Second, future studies should
employ different approaches to using salivary
biomarkers; that is, serial measurements may be more
valuable than one-point measurements. Further, a
panel of two or three salivary biomarkers may be
more sensitive than solitary biomarkers. Plus, many of
these biomarkers are not specific to CVD and are gen-
erally elevated in conditions associated with oxidative
stress or inflammation; therefore, a panel of suitable
biomarkers may have better specificity in detecting
CVD. In addition, future studies should employ a
large sample size and long follow-up periods to also
determine the prognostic value of these parameters.
Another recommendation for upcoming studies is to
determine accurate cutoff points for these biomarkers
to diagnose CVD.
Our review has some limitations, including the nar-
rative nature of the review that many aggravate some
source of bias (e.g., selection bias); however, we con-
ducted a systematic search to ensure including all
available studies eligible to our criteria. Another limi-
tation is the lack of high-quality, well-designed studies.
Moreover, there are not enough data regarding the
role of salivary biomarkers in risk stratification and
monitoring of therapy response. In addition, we could
not identify any reliable data in terms of novel
biomarkers discovered by omics approaches and
microbiome.
Conclusion
The clinical utility of salivary biomarkers to diagnose
CVD is promising but is still in the early stage of
development. Although some salivary biomarkers as
CRP, CK-MB, MYO, and TnI showed significant pro-
mise in diagnosing CVD, further studies are needed to
validate these findings, determine cutoff values for
diagnosis, and compare them to other established
biomarkers, currently in clinical use.
Acknowledgements
None.
Conflict of interest
The authors declare no conflict of interest.
Author contributions
EIB performed the data collection, searching data-
bases, initial draft writing, and final draft writing. CN
involved in conceptualization, critical review, and final
12
E. I. Bahbah et al.
draft writing. WP involved in initial draft writing and
final draft writing. MJ involved in visualization and
final draft writing. AW performed the conceptualiza-
tion, critical review, final draft writing, and submis-
sion.
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