ORIGINAL PAPERS

ORIGINAL PAPERS
Dent. Med. Probl. 2008, 45, 1, 33–36
ISSN 1644−387X
© Copyright by Silesian Piasts University of Medicine in Wrocław
and Polish Stomatological Association
ANETTA GMYREK−MARCINIAK, KATARZYNA HERMAN, URSZULA KACZMAREK
Salivary Flow Rate, Activity of Lysozyme and
Peroxidase in the Saliva of Children with Juvenile
Idiopathic Arthritis – Preliminary Study
Szybkość wydzielania śliny, aktywność lizozymu i peroksydazy
w ślinie dzieci z młodzieńczym idiopatycznym zapaleniem stawów
– doniesienie wstępne
Department of Conservative Dentistry and Pedodontics, Silesian Piasts University of Medicine in Wrocław, Poland
Abstract
Background. In juvenile idiopathic arthritis (JIA) patients some changes in saliva composition are observed.
Biochemical changes that may affect oral health were found in the saliva of patients suffering from juvenile idio−
pathic arthritis (JIA).
Objectives. The purpose of this study was to compare peroxidase and lysozyme levels of activity in mixed saliva
of children with JIA and control group.
Material and Methods. 137 children between 6 to 18 years of age were examined out 64 of which had JIA.
Unstimulated mixed saliva was collected. Salivary flow rate, lysozyme and peroxidase levels of activity were
assessed.
Results. Examination revealed lower salivary flow rate in JIA group than in the control group (0.21 ± 0.07 ml/min
vs. 0.58 ± 0.36 ml/min – flow rate). Significantly lower lysozyme activity in children with JIA in comparison to
control group was significantly.
Conclusions. Obtained results suggest JIA−related changes lower lysozyme activity in mixed saliva and lower sali−
vary flow rate (Dent Med. Probl. 2008, 45, 1, 33–36).
Key words: juvenile idiopathic arthritis, saliva, salivary flow rate, lisozyme, peroxidase.
Streszczenie
Wprowadzenie. U pacjentów chorych na młodzieńcze idiopatyczne zapalenie stawów (m.i.z.s.) obserwuje się
zmiany w składzie śliny. Biochemiczne zmiany zachodzące w ślinie pacjentów z młodzieńczym idiopatycznym za−
paleniem stawów (m.i.z.s.) mogą wpływać na zdrowie jamy ustnej.
Cel pracy. Porównanie poziomów aktywności peroksydazy i lizozymu śliny mieszanej dzieci chorujących na
m.i.z.s. oraz dzieci grupy kontrolnej.
Materiał i metody. Zbadano 137 dzieci w wieku 6–18 lat, w tym 64 dzieci chore na m.i.z.s. U każdego badanego
pobierano niestymulowaną ślinę mieszaną. Określano szybkość wydzielania śliny, poziom aktywności peroksyda−
zy oraz lizozymu śliny mieszanej.
Wyniki. Szybkość wydzielania śliny wynosiła u dzieci chorujących na m.i.z.s. 0,21 ± 0,07 ml/min, a w grupie kon−
trolnej 0,58 ± 0,36 ml/min. W ślinie mieszanej dzieci chorych na m.i.z.s. w porównaniu z grupą kontrolną stwier−
dzono istotnie niższy poziom aktywności lizozymu.
Wnioski. Uzyskane dane sugerują, związane z występowaniem m.i.z.s., zmiany poziomu aktywności lizozymu
w ślinie mieszanej oraz mniejsze szybkości wydzielania śliny (Dent Med. Probl. 2008, 45, 1, 33–36).
Słowa kluczowe: młodzieńcze idiopatyczne zapalenie stawów, ślina, szybkość wydzielania śliny, lizozym, pero−
ksydaza.
34
Juvenile arthritis disease encompasses many
forms. The most common form is juvenile
rheumatoid arthritis (JRA), also known as juvenile
idiopathic arthritis (JIA). All forms have a com−
mon manifestation of joint inflammation and an
onset before the age of 16, although they are
uncommon in the first six months of life. The dif−
ferent forms can be distinguished by specific
symptoms and complications that respond to par−
ticular treatments [1, 2]. The disease is a systemic
autoimmune disorder involving the release of
cytokines and matrix metalloproteinases from
inflammatory cells [3–5]. The inflammatory
process damages hard and soft tissues and causes
synovial hyperplasia and chronic inflammation.
Three major types of juvenile idiopatic arthri−
tis are known. Systemic JIA, which occurs in
about 10% of cases and causes joint pain,
swelling, fevers, and rash. The etiology of this
form of JIA is unknown. Polyarticular JIA occurs
in about 40% of cases and causes multiple painful,
swollen joints. Some children may have a positive
rheumatoid factor and the condition may turn into
rheumatoid arthritis. The etiology of this form of
JIA is also unknown. Pauciarticular JIA occurs in
about 50% of cases and is characterized by limited
involvement of joints. Some of the patients, in par−
ticular boys, are HLA−B27 positive. HLA−B27
marker was implicated in several autoimmune dis−
orders [1–3].
There are several areas where JIA may influ−
ence oral health, namely dental caries, periodontal
disease, saliva abnormalities, involvement of the
temporomandibular joint (TMJ) and the effect of
facial growth. In addition, the systemic effect of
chronic disease may also have impact on oral
health.
Saliva may be described as a heterogeneous
fluid composed of proteins, glycoproteins, elec−
trolytes, and small organic molecules as well as
compounds transported from blood [6, 7]. This
fluid constantly bathes the teeth and oral mucosa.
It acts as a cleansing solution, an ion reservoir,
a lubricant and a buffer [7]. Once saliva is present
at the tooth surface, the buffering action of saliva
may help to prevent further demineralization by
the plaque acid [7, 8]. Whole saliva represents
a mixture of the secretions of the major – sub−
mandibular, parotid, sublingual, and minor –
accessory salivary glands, together with the gingi−
val fluid [7]. The secretions from the different
glands have been shown to differ considerably, to
be complex in composition and to be affected by
different forms of stimulation, time of day, diet,
age, gender, a variety of disease states, and sever−
al pharmacological agents [9]. Salivary glands
contain many biochemical systems known to be
A. GMYREK−MARCINIAK, K. HERMAN, U. KACZMAREK
involved in soft tissue repair, and many antibacte−
rial components including lysozyme and salivary
peroxidase [7]. Salivary peroxidase plays an
important role in defense against invading
microorganisms and also limits accumulation of
toxic levels of hydrogen peroxide in the mouth
[10]. Lysozyme is a strong cationic cell−lysing
agent that can cause lysis of oral bacterial cells
[11]. Changes in saliva composition were
observed in patients diagnosed with juvenile idio−
pathic arthritis [12, 13].
The purpose of this study was to compare the
peroxidase and lysozyme activity in mixed saliva
of children with JIA to the healthy control.
Material and Methods
137 children, aged 6 to 18 years, were examined.
The study group included 64 children diagnosed with
JIA. The control group consisted of 73 healthy chil−
dren. Informed patient and parent consent and ethical
approval were obtained before the study.
Unstimulated whole saliva specimens were
obtained in the morning. Samples were stored on
ice before being frozen at –20°C while waiting for
laboratory analysis. The salivary secretion was
expressed as the volume of saliva (in ml) secreted
per minute.
Salivary peroxidase activity was measured
using the thionitrobenzoicacid (NBS) assay, as
described previously [12, 14]. Briefly, the colori−
metric change induced by the reaction between the
enzyme and the substrate (dithio−bis−2−nitroben−
zoic acid) in the presence of mercaptoethanol was
recorded at 412 nm for 20 s.
The activity of the lysozyme was measured by
the turbidimetric method described by Smolelis
and Hartsell in the Kopec’s modification. Briefly,
a turbid suspension of liofilizated culture
Micrococcus ATCC 4698 was used as a substrate.
The rate of the opacification under standard condi−
tions in the presence of saliva is a measure of
enzymatic activity. An absorbance was measured
at 620 nm. The enzyme concentration was deter−
mined using a standard curve.
The data were analyzed using t−Student test at
p < 0.05.
Results
The mean salivary flow rate value for the whole
group of patients with JIA was significantly lower
than in control group (0.21 ± 0.07 ml/min vs. 0.58
± 0.36 ml/min) in (Table 1). A similar finding was
obtained for lysozyme activity and measured by
35
Salivary Flow Rate, Activity of Lysozyme and Peroxidase in the Saliva of Children with JIA
Tabele 1. The comparison of component in saliva in children with JIA and control group
Tabela 1. Porównanie składników śliny u dzieci z m.i.z.s. oraz grupie kontrolnej
JIA group
(Grupa dzieci
chorych na m.i.z.s.)
Control group
(Grupa kontrolna)
Significant difference
(Istotność różnic)
N
x ± SD
N
x ± SD
Salivary flow rate – ml/min
(Szybkość wydzielania śliny – ml/min)
64
0.21 ± 0.07
73
0.58 ± 0.37
p < 0.05
Salivary peroxidase activity – IU/ml
(Aktywność peroksydazy – IU/ml)
64
0.83 ± 0.47
73
0.87 ± 0.53
p > 0.05
64
64
1.93 ± 1.83
7.35 ± 3.74
73
73
2.75 ± 2.81
10.53 ± 5.87
p < 0.05
p < 0.05
Salivary lysozyme activity – J/min
(Aktywność – lizozymu – J/min)
CETAB
H2O2
two methods [CETAB (1.93 ± 1.83 J/min vs. 2.75 ±
2.81 J/min) and H2O2 (7.35 ± 3.74 J/min vs. 10.53
± 5.87 J/min)] and presented in (Tab. 1). However,
salivary peroxidase activity was only slightly lower
in the studied group (0.83 ± 0.47 IU/ml vs. 0.87 ±
0.53 IU/ml) as indicated in (Table 1).
Discussion
Sjögren’s syndrome in children has seldom
been reported in association with JIA. Further
have, little was known about the involvement of
the salivary glands in the complications of JIA [15,
16]. Our study shows that the saliva flow rates
were reduced in patients suffering from JIA. It was
in agreement with previous studies by Walton et
al. [17]. Moen et al. [18] examined 50 patients
with rheumatoid arthritis, where 17 rheumatoid
arthritis patients were found to suffer from xeros−
tomia and 11 of them showed redused salivary
production. Of 33 patients that did not report
xerostomia, 9 were found to have salivary
hypoproduction. John et al. [19] showed 12–16%
of subjects had reduced salivary production.
Simapoloulou et al. [20] reported no difference in
parotid flow rates compared with controls.
Our study showed that the lysozyme activity
was also lower in the study group. This finding is
also in conformity with the previously published
work by Walton et al. [13].
Brik et al. [15] reported higher level of sali−
vary peroxidase activity in patients with JIA, par−
ticularly in the polyarticular group and the sys−
temic group (salivary peroxidase activity was
measured using the NBS assay). Nagler et al. [12]
also reported higher level of peroxidase activity in
rheumatoid arthritis patients. However, our study
differed from the published papers and did not
show a significant difference in peroxidase activi−
ty between the study and the control group.
The authors concluded that the salivary flow
rates were significantly lower in patients suffering
from JIA. The study showed significantly lower
lysozyme activity of saliva in children with JIA
than in control group. The salivary peroxidase
activity was only slightly lower in the studied
group. Obtained results suggest JIA−related
changes in lysozyme activity in mixed saliva and
lower salivary flow rate.
Acknowledgement. We are grateful to Dr. Maria Podwysocka−Harasimowicz for her assistance and fruitful discussions of this
work.
References
[1] FALCINI F., CIMAZ R.: Juvenile rheumatoid arthritis. Curr. Opin. Rheumatol. 2000, 12, 415–419.
[2] SOCHA J.: Choroby autoimmunologiczne u dzieci. Biblioteka Pediatry, Wydawnictwo Lekarskie PZWL, Warszawa
2005, 50, 93–97.
[3] ROMICKA A. M., ROSTROPOWICZ−DANISIEWICZ K.: Zapalne choroby reumatyczne w wieku rozwojowym.
Biblioteka Pediatry, Wydawnictwo Lekarskie PZWL, Warszawa 2005, 46, 71–87.
[4] HAVEMOSE−POULSEN A., WESTERGARD J., STOLTZE K., SKJØDT H., DANNESKIOLD-SAMSØE B., LOCHT H., BENDTZEN K.,
HOLMSTRUP P.: Periodontal and hematological characteristics associated with aggressive periodontitis, juvenile
idiopathic arthritis, and rheumatoid arthritis. J. Periodontol. 2006, 77, 280–288.
[5] BENDTZEN K.: Cytokines and natural regulators of cytokines. Immunol Lett. 1994, 43, 111–123.
[6] FDI Working Group 10, CORE: Saliva: its role in health and disease. Intern. Dent. J. 1992, 42, 291–304.
[7] BATTINO M., FERREIRO M. S., GALLARDO I., NEWMAN H. N., BULLON P.: The antioxidant capacity of saliva. J. Clin.
Periodontol. 2002, 29, 189–194.
36
A. GMYREK−MARCINIAK, K. HERMAN, U. KACZMAREK
[8] EDGAR W. M., HIGHAM S. M.: Saliva and the control of plaque. In: Saliva and control of plaque pH. Eds.: Edgar
W. M. and O’Mullane D. M., BDJ, London 1996, 2nd ed., 81–94.
[9] MANDEL I. D.: Relation of saliva and plaque to caries. J. Dent. Res. 1974, 53, 246–266.
[10] MANSSON−RAHEMTULLA B., BALDONE D. C., PRUITT K. M., RAHEMTULLA F.: Specific assays for peroxidases in
human saliva. Arch. Oral Biol. 1986, 31, 661–668.
[11] MANDEL I. D.: The role of saliva in maintaining oral homeostasis. JADA 1989, 119, 298–304.
[12] NAGLER R. M., SALAMEH F., REZNICK A. Z., LIVSHITS V., NAHIR A. M.: Salivary gland involvement in rheumatoid
arthritis and its relationship to induced oxidative stress. Rheumatology 2003, 42, 1234–1241.
[13] WALTON A. G., WELBURY R. R., THOMASON J. M., FOSTER H. E.: Oral health and juvenile idiopathic arthritis:
a review. Rheumatology 2000, 39, 550–555.
[14] REZNICK A. Z., KLEIN Y., EISFRICH J. P., CROSS E. C., NAGLER R.: Inhibition of oral peroxidase activity by ciga−
rette smoke: in vivo and in vitro studies. Free Rad. Biol. Med. 2003, 34, 377–384.
[15] BRIK R., LIVNAT G., POLLACK S., CATZ R., NAGLER R.: Salivary gland involvement and oxidative stress in juve−
nile idiopathic arthritis: novel observation in oligoarticular−type patients. J. Rheumatol. 2006, 33, 2532–2537.
[16] BARTUNKOVA J., SEDIVA A., VENCOVSKY J.: Primary Sjögren’s syndrome in children and adolescents: proposal for
diagnostic criteria. Clin. Exp. Rheumatol. 1999, 17, 381–386.
[17] WALTON A. G., WELBURY R. R., FOSTER H. E., WRIGHT W. G., THOMASON J. M.: Sialochemistry in juvenile idio−
pathic arthritis. Oral Dis. 2002, 8, 287–290.
[18] MOEN K., BERTELSEN L. T., HELLEM S., JONSSON R., BRUN J. G.: Salivary gland and temporomandibular joint
involvement in rheumatoid arthritis: relation to disease activity. Oral Dis. 2005, 11, 27–34.
[19] JOHN M., LAUERWALD A., JOHN V., THIEMANN H.H.: The production of saliva of patients with juvenile chronic
arthritis (JCA). Acta Universitatis Carolinae−Medica 1994, 40, 87–89.
[20] SIAMOPOULOUS A., MAVRIDIS A. K., VASAKOS S., BENECOS P., TZIOUFAS A. G., ANDONOPOULOS A. P.:
Sialochemistry in juvenile chronic arthritis. Br. J. Rheumatol. 1989, 28, 383–385.
Address for correspondence:
Anetta Gmyrek−Marciniak
Department of Conservative Dentistry and Pedodontics
Silesian Piasts University of Medicine
Krakowska 26
50−425 Wrocław
Poland
Tel.: +48 71 784 03 62
E−mail: [email protected]
Received: 7.04.2008
Revised: 28.04.2008
Accepted: 28.04.2008
Praca wpłynęła do Redakcji: 7.04.2008 r.
Po recenzji: 28.04.2008 r.
Zaakceptowano do druku: 28.04.2008 r.