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Available online at www.sciencedirect.com

SclenceDirect Procedio
Chemistrg
ELSEVIER Procedia Chemistry l8 (2016) 194 - 198

Molecular and Cellular Life Sciences: Infectious Diseases, Biochemistry and Structural Biology
2015 Conference, MCLS 2015

The Correlation between Pulmonary Function Tests and The

Salivary MMP-9 Activity among Chronic obstructive Pulmonary
Disease (COPD) Patients
Mutyadf*, Sunna#, Mulkan Azhary"
"Department of Pulmonolog ond Respiranry fu{edicfue, Facuily of lm:;Sliah ltuala IJaiv*sity, Kopelma Dorassalan, Battda Aceh 23 I t l,
h
Departmeat of Pertodontics, Faculty of batistry, Syiah Kuala IJniversity, Kopebna Darussolanq Banda Aceh 23 I I t , Inhnesia
"Department of Anoton4t and Histalog, Faculty of Medicine, Syiah Kualo lJniversiry, Kapelna Darussatom, Bando Aceh 231t l,Indonesia

Abstract

The spirometry test is routinely performed to assess FEVI, FVC, and FEVI/FVC ratio among chronic obskuctive
disease (COPD) patients with the increased activity of MMP-9. Saliva is less invasive to assess the MMp-9 a*ivity. This study
aimed to compare hrlmorary Function Tests lo estimate the MMP-9 activity.The respondents were 30 COpD outpatients from
Pulmonary Policlinic. Reults showed mean ratio of FEV,, FVC, FEV1/FVC (SD) aad that of the salivary MMp-9 activity were
1.67 (0.12) L,2.97 {0.43) L, 56'15 (8.43) % md 1.85 (1.54) pM The correlation between FEV,, FVC, and
FEVI/FVC ratio md Oe salivary MMP-9 activity was insipificant 1pX.OS1. TLe pulmonary function tests were not able to
estimate the salivary MMP-9 activity. The findings suggest further ac-tivitios of Lfil0-S fromother samples for comparison of
protease activity.

@ 2016 The Auth<rrs' Published by Elsevier B.V. This is an open asces article under the CC BY-NC-ND liceuse
(http:l/creativeoommons.orgAicenses/by-nc-nd/4.0/).
Peer-review under responsibility ofthe organizing committee of the Molecular and Cellular Life Sciences: Infectious Diseases,
Biochemistry and Smcnral Biolory 2015 (MCLS 20lS)
Keywordt : Spkometry; Salivary; MMP-9; Conrparison

+ Correspondirg author. T eL.:. t42-813-3557 -lS7 4

E-mai I a ddress : mri.O862(@gnail.com

1876-6196 @ 2016 TheAuthors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(htE://crativecommons.orglliceruedby-nc-nd/4.0/).
Peer-revisv under responsibility of the organizing committee of the Molecular and Cellular Life Sciences: Idectious Diseases, Biochemistry and
Structural Biology 2015 (MCLS 2015)
doi:l 0. l0l6/j.proche.2Ol6.0l.030
7

Mtlyadi et ol. / Prucedia Chemkry l8 (2016) 194 * 198 195

Broncho-alveolar lavage
Cbronic obstuctive pulmonary disease
Exhacellular matrix
Forced expiratory volume in I second
Forced vital capacity
Clobal initiative for chronic obstnrctive lung disease
Makix metalloproteinase-9
Tissue inhibitor of metalloproteinase- I

l. Introduction
The prevalence of chronic obstructive pulmonary disease (COPD) in Indonesia has reached 4.8 millions of
which 90% is smokers or former smokers. COPD is mainly triggered by smoke exposures and other dangerous
agants like gases and chemical substance from the environment The genetic inheritance, previous history of
respiratory infection, intrauterine growth retardation (IUGR), poor nutrition, and low income also contribute to the
increase of COPD. The passive smokers, approximately 2Ao/o, arc also risky for getting COPDL2.
Spirometry is routinely performed by those complaining dyspnoe promptly, so that they may have the
appopriate teatment subsequently. The calibrated spirometry is initially prepared before patients start the test.
Then, the measuremenb of forced vital capacity (FVC) and forced expiratory volume in one second (FEV1) me
performed with acceptable maneuvers. T.he results are very beneficial to determine either obstnrctiorl restriction, or
mixed. Regarding the diagnosis of COPD, tle baseline of GOLD 2013 is properly appliedr'2'3.
The increase of matrix metalloproteinase (MMP)-9 activity from plasma is correlated with o1-anti tripsin-related
emphyserna4s. Moreover, fie higho MMP-9 level is linem wi*r the lorryer FEV1, fransport of CO, and oxygen
saturation The increase of MMP-9 level might estimate the decline of pulmonary ftrnction and the risk of
exacerbation. The cigarette consumption is also correlated with MMP-9 lwela. Prior authors and colleagues have
started numsrous studies to detemine the correlation betwem different biological biomarkers and the pulmonary
fimction. For COPD, mauy biomarkers from broncho-alveolar lavage (BAL) fluid and sputum have been developed
in order to generate precise relationships between types ofbiomarkers and pulmonary function. Types of biomarliers
engaged in those studies were agents of oxidative stesg cytokines and vrious proteases describing the pathogenesis
of COPD. However, how to g$Jh" respiratory samples should be considered as well as those body fluids which
have not been easily obtaineds'6'7'8.
Since smoking would affect the pathological processes of oral cavity and lungs which induced inflammatory
mediators releasing MMP-9, our study mied to utilize the saliva to assess the MMP-9 activity from the fluid in order
to link the association between spirometry results and the activity of protease. Furthermore, hopefully, the
spirometry tests might be correlated adequately to the salivary MMP-9 activity among COPD patiarts.

2. Methods

This study employed thirty (30) smoker outpatients with COPD who visited pulmonary policlinic of Dr. Zainoel
Abidin Generxl Hospital in Banda Aceh - Indonesia. The sflrdy apptied cross sectional design to determine inclusion
and exclusion criteria for those emolled as rcspondents. In advance, they were confirmed as CSPD patients based
on sqiromehy results i.e. FEVIIFVC ratto <70Yo with mild, moderate, severe and very severe GOLD spirometric
level'. Other criteria were: smokers, male, >50 years old 20 pack years of cigarette consumption. The exclusion
criteria were hrberculosis and malignancy. The study was officially approved by the Ethics Committee of Faculty of
Medicine, Syiah Kuala University - Banda Aceh. All respoudents had to sign the informed consents before
undergoing the physical examination and saliva collection.
The saliva was collected by spitting out the fluid into the sterile pot. Respondants who had shown ttre worst oral
hygiene and bloody spittle discharge were excluded from the study. All collected saliva was stored in -80 0C freezer
for subsequent analysis with .Sersofute@ 520 Generic MMP assay kit Flaorimetric.
a

196 Mulyadi et al. / Prccedia Chemistry 18 (2016) 194 - Igt

3. Results and discussion

3.1. Chorqcteristics of respondents' spirometry test and salivary MMp-9 activity

This resemch erployed 30 respondents who were diagnosed with COPD regmdless GOLD obstruction criteria.
The spiromety tests showed mean ratio of FEV1, FVC, and FEV/FVC (SD) i.e. 1.67 (0.12) L,2.97 (0.43) L and
56.15 (8.43) 7o respectively. Moreover, the salivary MMP-9 activity was 1.85 (1.54) pM. Spiromety results showed
that FEVI/FVC ratio was <70% confirming that respondents suffered from COPD. Data were shown in Table l.

Table 1. Characteristics ofrespondeat's spirometry test and salivary MMp-g activity

FEVr G) 30 r.67 (0.r2)

rvcG) 30 7.97 {0.43)

FEVI/FVQ p1i6 (o7o; 30 56.r5 (8.43)

Safivary MMP-9 activity ftrM) 30 l.8s (1.54)

3-2. correlation bdween FEyt, Fyc, FEYt/rvc ratio and the salivary MMP-9 activity

The salivary MMP-9 activity was correlated with spirometry tests by using Pearson correlation test. A1l
spirometry values showed lower conelation towards the salivary MMP-9 activity with insignificant p values
(p>0.05) as shown in Table 2. The pulmonary fimction tesB might not conclude predicted enzyme activity of Ulp-
9.

Table 2. Cmrelation of FEV,, FVC, md FEVr/FVC ratio towards salivary MMp-9 activity

SaliY'ey MMP-9 activity

p value

FEVr (L) 0.119 0.s32

Fvc (L) 0.0t8 4.923

FEVr/FVC ratio (70) 0.161 0.396

3.3. Discussian

Smokers with COPD undervyent a decrease in FEVr and FVC, faster than non smokers, md so did the age
accelerating tle decrease concurrently. FEV1AVC ratio is one of tlre considerations to initially recogrrize tlre
obstructive diseases to determine whether CoPlsuspected patients should be teated as suffering iiom obstructive
lung disease^ or not. The estimated vahre of FEVI/FVC ratio should be confirmed and -determined more
appropriatelus" Our study revealed spiromety results accordingly to diapose COPD accurately (Table l).
Inflammatory progression on respiratory tracts, induced by smokingixposure, ld to the reliase of nurnerous
inflammatory cells predominantly nzuhorphils and macrophages that subsequently generated MMp-g. Release of
MMP-9 was also led by some cytokines and oxidative stress. Beside increased level oiMMP-9, the balance between
MMP-9 and tissue iuhibitor of metalloproteinase (TIMP)-1 also influenced the degradation since TIMP-I was the
main anti protease maintaining the balance with MMP-9 in order to prevent action of active MMp-g. According to
7

Mulyadi et al. / Pro*dia Chemisty l8 (2016) 194 - l,98 t97

spircmetry values, the degllne of FEV1 was linear with obstuction of airway caurcd by keakage of lung
exhacellular matix (ECM)4'10.
From periodontal tissue, periodontitis had more activity ofprotease, particularly MMP-9, released by neutophils
and fibroblasts toge&er with some cytokines e.g. IL-6 aud IL-10. Similar to what occured in lung tissues, increased
activity of MMP-9 and MMP-9ITIMP-I ratio would destruct ex&acellular matrix which finally caused
periodontitisl 1'12'r3.
This study analyzed the salivary MMP'9 activity as the saliva was easily collected with a sinrple proce&re.
hevious studies have assessed the level and agtivity of MMP-9 from various body fluids, mostly sputum,
bronchoalveolar lavage (BAL) and blood serum5'6'7. Increased actiyity of MMP-9 may cleave ECM of lung ti.suer.
Normally, those suffering from CI)PD, with lower FEV1 and FEV1/FVC ratio <70Yo, would also exhibit the
increased level and activity of MMP-9. However, the activity assessment was more appropriate as such activity
might represent the breakage of ECM than might the level of MMP-9. Instead of both pro MMP-9 and bond MMP-
9, the activity only measured active MMP-9r4.
The tide of MMP-9 activity apparently showed a dynamic sequence. Heal&y smokers would exhibit MMP-9
activity which was insignificant as compared to smokers with COPD. Frolonged smoking exposure was supposed to
be responsible upon MMP-9 activity. Moreover, TIMP-I also appemed to maintain the balance. Increased activity of
MMP-9 corresponded with fte airway obstruction and the deskuction of the exhacellulm matrix among COPD
patients. Moreover, the airway obstruction occured along with the increase of MMP-9/TIMP-I ratioe.
Spirometry also displayed dynamic values of pulmonary firnction test among COPD patients and so did MMP-9
activity. Our study was initialized with determining correlation between spirometry value and MMP-9 activity.
Unforhrnately, FEVI' FVC, and FEVTIFVC ratio correlated poorly and was insigrrificant (p>0.05) with salivary
MMP-9 activity (Table 2). The protease activity of MMP-9 apparently had its individual characteristics despik its
dpamical sequence. Smoken mostly from cigarettes, e,rtered through the oral cavity. This smoke, then, went into
lunp, yet the saliva was apparent to correspond better to the processes of the oral cavity diseaseslz,l3. The genetic
factor was supposed to affect the variability of MMP-9 activity ilnong COFD respondeuts. The genetic
susceptibility would be responsible to exprcss responsc of MMP-9 activity towmds expo$res, particularly smoking
exposure. In addition, role of TIMP-l also facilitated the feedback toward MMP-9 activityrs,
Due to inadequately comparable results of spirometry values towards salivary MMP-9 activity, this study
proposed to discover other MMP-9 activities from related biological samples. In this regards, we might suggest
further evaluation on MMP-9 activity from sputum (preferably) and BAL. Then, various activities of MMP-9 would
be compared to each other. The results would be valuable to estimate degradation of ECM from lrrng parenchyma
among COPD patients derived from some associated biological fluiitt''q?.
-
4. Conclusions

Since FEV1, FVC, and FEVI/FVC ratio showed insignificant p value towards the salivary MIVIP-9 activity
(p>0.05), pulmonary firnction tests apparently were poorly correlated with the salivary MMP-9 activity despite the
similar exposure, i.e. smoking. In the future, similar studies should deepen included criteria for those suitably gained
from many phenotlryes of COPD patients iu order to assess and estimate destuction of hmg parenchyma caused by
increased activity of MMP-9 iu accord with spiromety's FEVI, FVC, aud FEVTIFVC ratio.

Acknowledgements

We are very gratefirl to the Indouesian Ministry of Research-Technolory and Higher Education and Syiah Kuala
University for 2015 Fundamental Research Grant Batch l.

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