original papers - Advances in Clinical and Experimental Medicine

original papers - Advances in Clinical and Experimental Medicine

orIginal papers
Adv Clin Exp Med 2013, 22, 5, 659–666
ISSN 1899–5276
© Copyright by Wroclaw Medical University
Anna Zalewska1, A–D, Małgorzata Knaś2, A–D, Marek Niczyporuk3, B, C, E,
Hady Hady Razak4, B, C, E, Napoleon Waszkiewicz5, B, C, E, G,
Adrian Wojciech Przystupa6, B, C, E, Danuta Waszkiel1, E, F, Wiesław Zarzycki7, A, F
Salivary Lysosomal Exoglycosidases Profiles in Patients
with Insulin-Dependent and Noninsulin-Dependent
Diabetes Mellitus
Profil ślinowych egzoglikozydaz lizosomalnych
u pacjentów chorych na cukrzycę insulino- i nieinsulinozależną
Department of Conservative Dentistry, Medical University in Białystok, Poland
The Institute of Health Care, The Higher Vocational School – Prof. E.F. Szczepanik, Suwałki, Poland
3
Research Laboratory of Esthetic Medicine, Medical University in Białystok, Poland
4
1st Clinical Department of General and Endocrine Surgery, Medical University in Białystok, Poland
5
Department of Psychiatry, Medical University in Bialystok, Choroszcz, Poland
6
Maternity and Gynaecological Private Hospital in Białystok, Poland
7
Department of Endocrinology, Diabetology and Internal Disease, Medical University in Białystok,
Poland
1
2
A – research concept and design; B – collection and/or assembly of data; C – data analysis and interpretation;
D – writing the article; E – critical revision of the article; F – final approval of article; G – other
Abstract
Background. In this study we have investigated the effects of type I (insulin-dependent) and II (non-insulin
dependent) diabetes mellitus on the specific activity and the output of salivary exoglycosidases: N-acetyl-β-hexosoaminidase (HEX), and its isoenzymes A and B (HEX A, HEX B), and β-glucuronidase (GLU) in well controlled diabetic patients compared to healthy age-matched controls.
Material and Methods. In the saliva HEX, HEX A, HEX B and GLU were determined according to Marciniak et al.
Protein was determined by the Lowry et al. method.
Results. Our results show that in the case of type I diabetes, the significantly increased activity of salivary total HEX
is mainly due to the significantly increased HEX A specific activity. Significantly increased HEX specific activity
in DM II is an outcome of significantly increased HEX A as well as HEX B specific activities in comparison to the
appropriate healthy control. Our results showed a significant increase in the specific activity of GLU in saliva of
type II diabetes patients. The output of lysosomal exoglycosidases showed a similar significant increase compared
to the healthy control, in both groups of diabetes mellitus patients.
Conclusions. This study has demonstrated that non-insulin dependent diabetes mellitus more strongly modify salivary glands glycoconjugates catabolism, which can be attributed to functional and morphological changes. A significant increase in the outputs of exoglycosidases in saliva of both type diabetes patients once more indicates that
special attention should be paid to the oral health of these patients (Adv Clin Exp Med 2013, 22, 5, 659–666).
Key words: diabetes mellitus, saliva, exoglycosidases.
Streszczenie
Wprowadzenie. W badaniach porównano wpływ cukrzycy I (insulinozależnej) i II (insulinoniezależnej) typu na
aktywność specyficzną i wydzielanie ślinowych egzoglikozydaz: N-acetylo-β-heksozoaminidazy (HEX) i jej izoenzymów A i B (HEX A i HEX B) oraz β-glukuronidazy (GLU) w ślinie pacjentów z uregulowaną cukrzycą typu I i II
w porównaniu z dobraną pod względem wieku grupą kontrolną.
660
A. Zalewska et al.
Materiał i metody. Aktywność specyficzną HEX, HEX A, HEX B i GLU w ślinie oznaczono metodą Marciniak et al.,
a stężenie białka metodą Lowry et al.
Wyniki. Wykazano, że w przypadku cukrzycy typu I istotnie zwiększyła się aktywność HEX w ślinie całkowitej,
głównie ze względu na istotne zwiększenie aktywności specyficznej HEX A. Istotne zwiększenie aktywności specyficznej HEX w cukrzycy typu II jest wynikiem zarówno istotnego zwiększenia HEX A, jak i HEX B w stosunku do badań kontrolnych. Wykazano także istotne zwiększenie aktywności specyficznej GLU w ślinie pacjentów
z cukrzycą typu II. Zaobserwowano podobne zwiększenie wydzielania wszystkich badanych egzoglikozydaz ślinowych w obu typach cukrzycy.
Wnioski. Badanie wykazało, że cukrzyca typu II silniej modyfikuje katabolizm glikokoniugatów w gruczołach ślinowych, co można tłumaczyć zmianami czynnościowymi i morfologicznymi zachodzącymi w śliniankach. Znaczące
zwiększenie wydzielania egzoglikozydaz w ślinie pacjentów z cukrzycą obu typów wskazuje na konieczność poświęcenia szczególnej uwagi na zdrowie jamy ustnej tej grupy pacjentów (Adv Clin Exp Med 2013, 22, 5, 659–666).
Słowa kluczowe: cukrzyca, ślina, egzoglikozydazy.
Several systemic diseases, e.g. rheumatoid arthritis [1], sclerodermia [2], systemic lupus erythematous [2], and diabetes [3] influence the salivary glands morphology and function. It was
reported that diabetes type I (insulin-dependent)
and II (non-insulin dependent) are the most common metabolic diseases with salivary glands complication [4]. The diabetes sialosis is an outcome
of lipid infiltration of the salivary glands, decreasing the size and the number of acinar cells and salivary ducts and the presence of immune complexes similar to the one occurring in the pancreas of
diabetic patients [4]. The functional changes in diabetic salivary glands were also described: altered
secretory protein expression, decreased response
to autonomic stimulation, increased autophagy and lysosomal activity and the accumulation
of basement membrane material [5]. The diabetes sialosis, similar to other degenerative diseases,
is a multifactor biochemical and immunological
process that is due in part to the action of several
enzymes [6].
N-acetyl-β-hexosoaminidase (EC 3.2.1.52,
HEX) hydrolyses oligosaccharide chains of glycoconjugates and it is the most active lysosomal exoglycosidase. In tissue and body fluids isoenzymes A (subunit α, β), B (subunit β, β), P, I, C and
S (subunit α, α) have been detected. Normal body
fluids are dominated by isoenzyme A (HEX A) and
B (HEX B) activity. HEX A is a part of a soluble intralysosomal component and may reflect the secretory activity of the cell. HEX B is bound with the cellular membrane and reflects an impaired membrane
function and damage of the cells [7]. β-glucuronidase
(GLU) is an enzyme responsible for the degradation
of proteoglycans and the ground substance [8] and it
is stored mainly in primary granules of polymorphonuclear leukocytes (PMN) [9].
Salivary exoglycosidases are a group of enzymes that in concert are responsible for the degradation of oligosaccharide chains of glycoconjugates
during organogenesis, growth and normal tissue
turnover. Glycoconjugates include glycoproteins
and glycolipids, constituting cellular membranes,
e.g. salivary glands cells as well as polysaccharide
chains of glycosaminoglycans and proteoglycans
constituting with glycoproteins extracellular matrix [10]. Glycoconjugates catabolism depends on
the continuous renewal of cellular elements of the
salivary glands; it is started by endoglycosidases
and then successively cleaved at the non-reducing
end of oligosaccharide chains by exoglycosidases [10]. The activity and the output of exoglycosidases in saliva is normally quite low. The elevation
in the specific activities of salivary exoglycosidases may be proof of losing balance between degradation of older and synthesis of newest elements,
which is observed in different pathological states of
the salivary glands. Bierć et al. [11] noted a significant increase in activities of β glucuronidase, β galactosidase, and HEX with its isoenzymes in the
salivary glands tumors. Waszkiewicz et al. [12] observed that the large single dose of ethanol significantly elevated the specific activity of salivary HEX
and its isoenzymes. Knaś et al. [3] showed that diabetes type I in smoking individuals significantly
increases salivary total HEX specific activity.
The true effects of diabetes in the catabolism
of salivary glycoconjugates of well-controlled patients and the way that the two types of the diabetes affect the salivary glands are not explained.
In this study we have investigated the effects
of type I and II diabetes mellitus on the specific
activity and the output of salivary exoglycosidases: HEX, HEX A, HEX B, and GLU in well controlled diabetic patients compared to healthy agematched control.
Material and Methods
60 diabetic patients and 60 healthy participants
were qualified into this study. The study was conducted on diabetic patients treated in the Department of Endocrinology, Diabetology and Internal
Disease, Medical University in Bialystok, Poland.
Written informed consent was obtained from each
661
Salivary Exoglycosidases in Type I nad II Diabetes
participant after the aims and methodology of the
study were explained. The study was approved by
the Ethics Committee of the Medical University of
Bialystok (permission number R-I-002/226/2006).
Patients and healthy participant were excluded if
they used medication which could cause salivary
glands hypofunction. None of the patients had
rheumatoid arthritis, sclerodermia or hypertension; patients with HCV and HIV infection were
also not included. None of the participant was
smokers. A check-up of the oral cavity was done
by the same dentist in artificial light using diagnostic dental tools (dental mirror and probe) following WHO criteria [13]. The dental status in all participants was determined by DMFT index (Decay,
Missing, Filled Teeth), gingival status was examined by gingival index (GI, a score 0–3) and sulcus
bleeding index (SBI, a score 0–4), the periodontal
status was evaluated by probing depth (PD).
Participants were divided into four study
groups, each consisting of 30 persons (15 women,
15 men):
1)  control for type I diabetes – the first control
(C I) – mean age 29 ± 6 years; DMFT 15 ± 3; GI
0.58 ± 0.05; SBI (%) 7.0 ± 0.8; PD 2.5 ± 0.2 mm;
2)  diabetes mellitus type I (DM I) – mean age
29 ± 5 years; disease duration 12.12 ± 2.51 years;
BMI 24.36 ± 4.59; preprandial glycaemia 124.6 ±
± 4.78 mg/dL; postprandial glycaemia 136.81 ±
± 5.15 mg/dL; glycaemia during sample collection 139.15 ± 5.31 mg/dL; HbA1C 6.91 ± 0.32%;
DMFT 13 ± 4, GI 0.52 ± 0.08, SBI (%) 8.0 ± 0.35,
PD 2.6 ± 0.15 mm;
3)  control for type II diabetes – the second control (C II) – mean age 54 ± 3 years; DMFT 12 ± 2; GI 0.64 ± 0.08; SBI (%) 9.0 ± 0.6; PD
2.8 ± 0.5 mm;
4)  diabetes mellitus type II (DM II) – mean age
59.39 ± 6.4 years; disease duration 9.9 ± 1.07 years;
BMI 30.45 ± 5.86; preprandial glycaemia 124.72 ±
± 4.79 mg/dL; postprandial glycaemia 150.5 ±
± 5.67 mg/dL, glycaemia during sample collection
134.97 ± 5.22 mg/dL; HbA1C 7.06% ± 0.31; DMFT 14 ± 3; GI 0.58 ± 0.09; SBI (%) 8.0 ± 0.4; PD
2.7 ± 0.8 mm.
Unstimulated
Whole Saliva Collection
Participants were instructed to refrain from
food and beverages, except water, for two hour
before their saliva was to be collected. All salivary samples were collected by the same person
at a fixed time of the day, in this study between
8 a.m. and 10 a.m., in order to minimize fluctuations related to a circadian rhythm of salivary secretion and composition. During saliva collection
the patient was seated on a chair and protected
from any stimulation. The whole saliva was collected in a plastic tube placed on ice by the spitting
method under standardized conditions [14]. The
secretion rate of unstimulated whole saliva was
measured during the time required to obtain 5 mL
of saliva. Immediately after collection, the salivary
samples were centrifuged at 3,000 × g for 20 minutes at 4oC to remove cells and debris. The resulting supernatants were divided into 50 µL portions,
frozen and kept at –800C until analyzed.
Analytical Methods
As a substrate for determination of the HEX
activity, 4-nitrophenyl-N-acetyl-β-glucosaminide
(Sigma, St. Louis, MO, USA) was used, as well as
p-nitrophenyl-β-D-glucuronide (Fluka Chemie;
Sigma, St. Louis, MO, USA) for GLU. Activities of
total HEX as well as GLU, in supernatants of saliva
were determined in duplicates according to Marciniak et al. [8]. HEX A specific activity was calculated as the difference between the total HEX and
HEX B activity.
Spectrophotometric measurements of p-nitrophenol released by the exoglycosidases activity
was carried out at 405 nm using microplate reader Elx800TM. Protein content in saliva was determined in duplicate by Lowry method [14].
Statistics
Statistical analyses were done using Statistica
10.0 (StatSoft, Cracov, Poland) according to ANOVA and post hoc test. Pearson’s correlation coefficient was used to determine the association between two variables. Results were expressed as
means ± SD. Statistical significance was defined as
p < 0.05.
Results
The specific activity of salivary HEX in DM
I group (600.99 ± 64.91 pKat/kg of protein) was
1.3 times significantly increased in comparison
to the appropriate control (C I) (450.63 ± 234.58
pKat/kg of protein). The specific activity of salivary HEX in DM II group (1130.81 ± 504.76 pKat/
/kg of protein) was 2.6 times higher in comparison to the control (C II) (439.48 ± 109.51 pKat/
/kg of protein) (Fig. 1A). The output of salivary HEX: in DM I group (238.5 ± 115.96 pKat/
min) and DM II group (497.49 ± 188.65 pKat/
min) was significantly 1.5 times higher in comparison to C I (163.48 ± 90.00 pKat/min) and C II
(327.44 ± 114.8 pKat/min) (Fig. 2A).
662
A. Zalewska et al.
Fig. 1. The specific activity of salivary lysosomal exoglycosidases in DM I and II group in comparison to the
appropriate control. HEX – N-acetyl-β-hexosoaminidase, HEX A – isoenzyme A of N-acetyl-β-hexosoaminidase,
HEX B – isoenzyme B of N-acetyl-β-hexosoaminidase, GLU – β-glucuronidase, C I – control for type I diabetes,
DM I – diabetes mellitus type I, C II – control for type II diabetes, DM II – diabetes mellitus type II, ↑* – significant
increase, ↑ – non-significant increase
Ryc. 1. Aktywność specyficzna ślinowych egzoglikozydaz lizosomalnych w cukrzycy typu I i II w porównaniu
z odpowiednią grupą kontrolną. HEX – N-acetylo-β-heksozoaminidaza, HEX A – izoenzym A N-acetylo-β-heksozoaminidazy, HEX B – izoenzym B N-acetylo-β-heksozoaminidazy, GLU-β-glukuronidaza, C I – badanie kontrolne
I typu cukrzycy, DM I – cukrzyca I typu, C II – badanie kontrolne II typu cukrzycy, DM II – cukrzyca II typu,
↑* – wzrost istotny statystycznie, ↑ – wzrost nieistotny statystycznie
The specific activity of salivary HEX A in
DM I group (285.61 ± 93.8 pKat/kg of protein)
was 1.6 times significantly higher in comparison
to the appropriate control (C I) (171.53 ± 132.52
pKat/kg of protein). The specific activity of salivary HEX A in DM II group (616.49 ±528.66 pKat/
/kg of protein) was 2.9 times higher in comparison to the control (C II) (207.79 ± 104.71 pKat/
/kg of protein) (Fig. 1B). The output of salivary
HEX A: in DM I group (147.29 ± 97.3 pKat/min)
and in DM II group (269.48 ± 162.99 pKat/min)
was significantly 1.5 and 1.8 times higher in comparison to C I (94.6 ± 72.98 pKat/min) and C II
(123.85 ± 77.63 pKat/min), respectively (Fig. 2B).
The specific activity of salivary HEX B in
DM I group (315.38 ± 86.19 pKat/kg of protein)
showed strong tendency to increase in comparison to the control group (271.1 ± 112.15 pKat/
/kg of protein). The specific activity of salivary
HEX B in DM II group (534.6 ± 135.21 pKat/kg
of protein) was 2.3 times higher in comparison
to the appropriate control (231.69 ± 60.92 pKat/
/kg of protein) (Fig. 1C). The output of salivary
HEX B in: DM I (91.2 ± 32.09 pKat/min) and in
DM II (228.01 ± 64.83 pKat/min) was significantly 1.3 times higher in comparison to the appropriate control: C I (68.88 ± 24.79 pKat/min) and C II
(180.89 ± 59.61 pKat/min) (Fig. 2C).
The specific activity of salivary GLU in DM
I group (117.57 ± 14.17 pKat/kg of protein) showed
strong tendency to increase in comparison to the
appropriate control C I (110.00 ± 32.82 pKat/kg
663
Salivary Exoglycosidases in Type I nad II Diabetes
Fig. 2. The output of salivary lysosomal exoglycosidases in DM I and II group in comparison to the appropriate control. HEX – N-acetyl-β-hexosoaminidase, HEX A – isoenzyme A of N-acetyl-β-hexosoaminidase, HEX B – isoenzyme
B of N-acetyl-β-hexosoaminidase, GLU – β-glucuronidase, C I – control for type I diabetes, DM I – diabetes mellitus
type I, C II – control for type II diabetes, DM II – diabetes mellitus type II, ↑* – significant increase, ↑ – non-significant increase.
Ryc. 2. Wydzielanie ślinowych egzoglikozydaz lizosomalnych w cukrzycy typu I i II w porównaniu z odpowiednią
grupą kontrolną. HEX – N-acetylo-β-heksozoaminidaza, HEX A – izoenzym A N-acetylo-β-hekso-zoaminidazy,
HEX B – izoenzym B N-acetylo-β-heksozoaminidazy, GLU-β-glukuronidaza, C I – badanie kontrolne I typu cukrzycy,
DM I – cukrzyca I typu, C II – badanie kontrolne II typu cukrzycy, DM II – cukrzyca II typu, ↑* – wzrost istotny
statystycznie, ↑ – wzrost nieistotny statystycznie
of protein). The specific activity of salivary GLU
in DM II group (171.07 ± 76.68 pKat/kg of protein) was significantly 3.1 times higher in comparison to the appropriate control (55.16 ± 15.09
pKat/kg of protein) (Fig. 1D). The output of salivary GLU: in DM I group (102.77 ± 48.18 pKat/
/min) and in DM II group (70.14 ± 13.91 pKat/
/min) was significantly 1.5 times higher in comparison to C I (70.72 ± 32.49 pKat/min) and C II
(52.20 ± 30.09 pKat/min), respectively (Fig. 2D).
There were no correlations between disease
duration, BMI, preprandial glycaemia, postprandial glycaemia, glycaemia during sample collection,
HbA1C and the specific activities of exoglycosidases examined.
Disscussion
The present study for the first time demonstrates that the salivary glands glycoconjugates and
salivary glycoproteins catabolism, measured by the
specific activity and the output of the lysosomal exoglycosidases secreted into saliva, is changed
and it is more disturbed in type II diabetes mellitus
in comparison to type I diabetes mellitus. These
changes in salivary exoglycosidases activites may
be related with changes in the ultrastructure and
function of the salivary glands, the changes in the
outputs of salivary exoglycosidases may facilitate
the development of oral diseases.
Our results show that in the case of type I diabetes, the significantly increased activity of salivary
664
total HEX is mainly due to the significantly increased HEX A specific activity and in less extent
due to the moderate tendency to increase in HEX B specific activity. Significantly increased HEX specific activity in DM II is an outcome of significantly increased HEX A as well as HEX B specific activities in comparison to the appropriate healthy
control (Figs. 1A, 1B, 1C).
It has been found that the specific activity of
salivary total HEX increases during salivary gland
dysfunction [3, 12]. However, a mechanism responsible for increasing total HEX and its isoenzymes specific activity outside the cell- in saliva is
not explained. The permeabilisation of lysosomal
membrane and leakage of the enzymes via cytosol to the saliva by increased basement membrane
permeability and/or cell membrane damage, enhanced synthesis or delayed removal of these enzymes by activated reticuloendothelial cells may be
considered as a reason of increased salivary HEX
and its isoenzymes activity in body fluids [7, 10].
As the activity of HEX A reflects the secretory
status of the cell [7], the significant increase of HEX A in saliva in type I and II diabetes mellitus patients
suggests that increased production of HEX A is related to functional changes in salivary glands
cells. The activity of HEX B is an outcome of the
breakdown of the cells membrane [7]. Since it was
shown in experimentally induced diabetes that the
changes in the ultrastructure of the submandibular glands (e.g. damaged of mitochondria, swollen
lysosomes, accumulation of large amounts of lipids
in the intracellular spaces) were correlated with the
increased HEX specific activity [15], so we hypothesize that a significant increase in HEX B specific
activity in saliva of type II diabetes mellitus may be
an indicator of an advanced pathological changes in
the cell structure of the salivary glands of a human.
A moderate tendency to increase the HEX B specific activity in saliva of type I diabetes might suggest
a less intensive degeneration of the salivary gland
parenchyma during insulin dependent diabetes.
PMN are the key cells of the innate immunity
system and belong to the first cells at the site of the
infection. The bactericidal activities of PMN include the generation of NO and reactive oxygen to
kill phagocytosed pathogens as well as the release
of a range of enzyme e.g. β-glucuronidase [16]. In
the present work, we showed a significant increase
in the specific activity of GLU in saliva of type II
diabetes patients and a strong tendency to increase
in the specific activity of GLU in saliva of type I diabetes patients, which may suggest the presence of
a local inflammatory state in the salivary glandular
cells, as salivary GLU is treated as a marker of neutrophils infiltration and a release of primary granules by neutrophils [17, 18].
A. Zalewska et al.
We believed that in the case of type I diabetes
mellitus functional changes in the salivary glands
cells dominate over ultrastructural changes. In the
case of type II diabetes both: damage and functional changes in the cells equally influence the salivary glands and they are more pronounced than
in the type I diabetes mellitus. The reason for this
phenomenon is not explained. We were not able
to find in the literature any data comparing the activity of lysosomal exoglycosidase in the saliva of
diabetic type I and type II, so it is impossible to
compare our results with the results of other authors. Our previous paper showed that diabetes
type I and smoking modify the activity of HEX and
its isoenzymes (similar trend to the present study
with the exception that HEX B specific activity in
saliva od diabetic patients showed very weak tendency to decrease in comparison to the control),
but only a combination of diabetes and smoking
significantly increases in the specific activity HEX
and its isoenzymes [3].
The oral cavity health depends on soluble salivary glycoproteins (19) as well as epithelial cells components [20] containing numerous
cell-membrane glycoproteins. Salivany glycoproteins are an important part of oral defense: they
take part in bacterial clearance and regulate bacterial colonization of oral tissue [21, 22] as well
as they are a part of innate and adaptive immune
systems [23, 24], provide fluid layers with highly effective lubricating properties [25] and maintain pH of saliva [26]. One of the important parts
in the degradation of oral glycoconjugates is deglycosylation [27], which depends on the removal
of the single monosaccharide from non-reducing
end of oligosaccharide chains by exoglycosidases
secreting, along with saliva, into the oral cavity.
An increase in the output means that more of the
catabolic enzyme is secreted into the saliva in one
minute of the saliva collection, which causes an
imbalance between the degradation of older and
the synthesis of the newest glycoconjugates. The
salivary outputs of exoglycosidases examined increased similarly in both groups (Fig. 2). Significantly more exoglycosidases in diabetic patients
saliva may accelerate the degradation of salivary
and pellicle glycoproteins as well as glycosaminoglycans of oral mucosa surfaces and result in oral
mucosa diseases, candidiasis, periodontitis, xerostomia and caries described by others in professional literature [28, 29].
In conclusion, this study has demonstrated
that non-insulin dependent diabetes mellitus more
strongly modify the salivary glands glycoconjugates catabolism, which can be attributed to functional and morphological changes. A significant
increase in the outputs of exoglycosidases in the
Salivary Exoglycosidases in Type I nad II Diabetes
saliva of both type diabetes patients once more indicates that special attention should be paid to the
oral health of these patients. Therefore, diabetic
665
patients require special attention of diabetologists
and special care of stomatologists.
References
  [1] Jensen JL, Uhlig T, Kvien TK, Axell T: Characteristics of rheumatoid arthritis patients with self- reported sicca
symptoms: evaluation of medical, salivary and oral parameters. Oral Diseases 1997, 3, 254–261.
  [2] Kalk WW, Vissink A, Spijkervet FK, Bootsma H, Kallemberg CG, Nieuw Amerongen AV: Sialometry and sialochemistry: diagnostic tools for Sjogren’s syndrome. Ann Rheum Dis 2001, 60, 1110–1116.
  [3] Knaś M, Karaszewska K, Szajda SD, Zarzycki W, Dudzik D, Zwierz K: Saliva of patients with Type I diabetes:
effect of smoking on activity of lysosomal exoglycosidases. Oral Dis 2006, 12, 278–282.
  [4] Mata AD, Marques D, Rocha S, Francisco H, Santos C, Mesquita MF, et al.: Effects of diabetes mellitus on salivary secretion and its composition in the human. Mol Cell Biochem 2004, 20, 1–6.
  [5] Mednieks MI, Szczepański A, Clark B, Hand AR: Protein expression in salivary glands of rats with streptozotocin
diabetes. Int J Exp Path 2009, 90, 412–422.
  [6] Chojnowska S, Kępka A, Szajda SD, Waszkiewicz N, Bierć M, Zwierz K: Exoglycosidase markers of diseases.
Biochem Soc Trans 2011, 39, 406–409.
  [7] Zwierz K, Zalewska A, Zoch-Zwierz W: Isoenzymes of N-acetyl-beta-hexosaminidase. Acta Biochim Polon 1999,
46, 739–757.
  [8] Marciniak J, Zalewska A, Popko J, Zwierz K: Optimization of an enzymatic method for the determination of lysosomal
N-acetyl-beta-D-hexosoaminidase and beta-glucuronidase in synovial fluid. Clin Chem Lab Med 2006, 44, 933–937.
  [9] Lamster IB, Harper DS, Fiorello LA, Osharin RL, Celenti RS, Gordon JM: Lysosomal and cytoplasmic enzyme
activity, cervicular fluid volume, and clinical parameters characterizing gingival sites with shallow to intermediate
probing depths. J Periodontol 1987, 58, 614–621.
[10] Zwierz K, Gindzieñski A, Ostrowska L, Stankiewicz-Choroszczucha B: Metabolism of glycoconjugates in human
gastric mucosa – a review. Acta Med Hung 1989, 46, 275–288.
[11] Bierć M, Minarowski Ł, Woźniak Ł, Chojnowska S, Knaś M, Szajda SD, et al.: The activity of selected glycosidases in salivary gland tumors. Folia Histochem Cytobiol 2010, 48, 471–474.
[12] Waszkiewicz N, Szajda SD, Jankowska A, Kępka A, Dobryniewski J, Szulc A, et al.: The effect of the binge
drinking session on the activity of salivary, serum, urinary beta Hexosaminidase: Preliminary data. Alcohol and
Alcoholism 2008, 43, 446–450.
[13] Navazesh M, Christensen C, Brightman V: Clinical criteria for the diagnosis of salivary gland hypofunction.
J Dent Res 1992, 71, 1363–1369.
[14] Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol
Chem 1951, 193, 265–275.
[15] Maciejewski R, Burdan F, Hermanowicz-Dryka T, Wójcik K, Wójtowicz Z: Changes in the activity of some lysosomal enzymes and in the fine structure of submandibular gland due to experimental diabetes. Act Physiol Hung
1999, 86, 127–137.
[16] Wessels I, Jansen J, Rink L, Uciechowski P: Immunosenescence of polymorphonuclear neutrophils. The Scientific
World J 2010, 10, 145–160.
[17] Albandar JM, Kingman A, Lamster IB: Cervicular fluid level of beta-glucuronidase in relation to clinical periodontal
parameters and putative periodontal pathogens in early – onset periodontitis. J Clin Periodontol 1998, 25, 630–639.
[18] Eley BM, Cox SW: Advances in periodontal diagnosis 7. Proteolytic and hydrolytic enzymes link with periodontitis. Brit Dent J 1999, 184, 323–328.
[19] Zalewska A, Zwierz K, Żółkowski K, Gindzieński A: Structure and biosynthesis of human salivary mucins. Acta
Biochim. Polon. 2000, 47, 1067–1079.
[20] Kleinberg I, Westbay G: Salivary and metabolic factors involved in oral malodor formation. J Periodontol 1992,
63, 768–775.
[21] Mandel ID: The functions of saliva. J Dent Res 1987, 66, 623–627.
[22] Mandel ID: The role of saliva in maintaining oral homeostasis. JADA 1989, 119, 298–304.
[23] Tenovuo J: Antimicrobial Agents in Saliva-Protection for the whole body. J Dent Res 2002, 81, 807–809.
[24] Tenovuo J, Pruiti KM: Relationship of the human salivary peroxidase system to oral health. J Oral Pathol 1984,
13, 573–584.
[25] Alliende C, Kwon YJ, Brito M, Molina C, Aguilera S, Perez P, et al.: Reduced sulfatation of muc5b is linked to
xerostomia in patients with Sjogren syndrome. Ann Rheum Dis 2009, 67, 1480–1487.
[26] Zalewska A, Pietruska MD, Knaś M, Zwierz K: Niemucynowe białka śliny o wysokim stopniu homologii łańcucha
polipeptydowego. Post Hig Med Dośw 2001, 55, 733–754.
[27] Gottschalk A, Fazekas De St Groth S: Studies on mucoproteins. III. The accessibility to trypsin of the susceptible
bond in ovine submaxillary gland mucoprotein. Biochim Biophys Acta 1960, 43, 513–519.
[28] Chi AC, Neville BW, Krayer JW, Gonsalves WC: Oral manifestations of systemic disease. Am Fam Physician
2010, 82, 1381–1388.
[29] Siudikiene J, Machiulskiene V, Nyvad B, Tenovuo J, Nedzelskiene I: Dental caries increments and related factors
in children with type 1 diabetes mellitus. Caries Res 2008, 42, 354–362.
666
A. Zalewska et al.
Address for correspondence:
Anna Zalewska
Marii Curie-Skłodowskiej 24a
15-275 Białystok
Poland
Tel.: +48 85 745 09 61
E-mail: [email protected]
Conflict of interest: None declared
Received: 4.06.2012
Revised: 12.07.2012
Accepted: 3.10.2013