Anna Zalewska
1, A–D, Małgorzata Knaś
2, A–D, Marek Niczyporuk
3, B, C, E,
Hady Hady Razak
4, B, C, E, Napoleon Waszkiewicz
5, B, C, E, G,
Adrian Wojciech Przystupa
6, B, C, E, Danuta Waszkiel
1, E, F, Wiesław Zarzycki
7, A, FSalivary 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
1 Department of Conservative Dentistry, Medical University in Białystok, Poland
2 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
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 con-trolled 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 sal-ivary glands glycoconjugates catabolism, which can be attributed to functional and morphological changes. A sig-nificant 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 izoen-zymów 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.
Adv Clin Exp Med 2013, 22, 5, 659–666 ISSN 1899–5276
ORIGINAL PAPERS
Several systemic diseases, e.g. rheumatoid ar-thritis [1], sclerodermia [2], systemic lupus ery-thematous [2], and diabetes [3] influence the sal-ivary glands morphology and function. It was reported that diabetes type I (insulin-dependent) and II (non-insulin dependent) are the most mon metabolic diseases with salivary glands com-plication [4]. The diabetes sialosis is an outcome of lipid infiltration of the salivary glands, decreas-ing the size and the number of acinar cells and sal-ivary ducts and the presence of immune complex-es similar to the one occurring in the pancreas of diabetic patients [4]. The functional changes in di-abetic salivary glands were also described: altered secretory protein expression, decreased response to autonomic stimulation, increased autopha-gy and lysosomal activity and the accumulation of basement membrane material [5]. The diabe-tes 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 gly-coconjugates and it is the most active lysosom-al exoglycosidase. In tissue and body fluids isoen-zymes 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 in-tralysosomal component and may reflect the secre-tory activity of the cell. HEX B is bound with the cel-lular 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 polymorpho-nuclear leukocytes (PMN) [9].
Salivary exoglycosidases are a group of en-zymes that in concert are responsible for the degra-dation 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 ma-trix [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 exoglycosidas-es [10]. The activity and the output of exoglycosi-dases in saliva is normally quite low. The elevation in the specific activities of salivary exoglycosidas-es may be proof of losing balance between degra-dation of older and synthesis of newest elements, which is observed in different pathological states of the salivary glands. Bierć et al. [11] noted a signifi-cant increase in activities of β glucuronidase, β ga-lactosidase, and HEX with its isoenzymes in the salivary glands tumors. Waszkiewicz et al. [12] ob-served that the large single dose of ethanol signifi-cantly elevated the specific activity of salivary HEX and its isoenzymes. Knaś et al. [3] showed that di-abetes 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 pa-tients and the way that the two types of the diabe-tes 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 exoglycosidas-es: HEX, HEX A, HEX B, and GLU in well con-trolled diabetic patients compared to healthy age-matched control.
Material and Methods
60 diabetic patients and 60 healthy participants were qualified into this study. The study was con-ducted on diabetic patients treated in the Depart-ment of Endocrinology, Diabetology and Internal Disease, Medical University in Bialystok, Poland. Written informed consent was obtained from each
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 spe-cyficznej HEX w cukrzycy typu II jest wynikiem zarówno istotnego zwiększenia HEX A, jak i HEX B w stosun-ku do badań kontrolnych. Wykazano także istotne zwiększenie aktywności specyficznej GLU w ślinie pacjentów z cukrzycą typu II. Zaobserwowano podobne zwiększenie wydzielania wszystkich badanych egzoglikozydaz ślino-wych w obu typach cukrzycy.
Wnioski. Badanie wykazało, że cukrzyca typu II silniej modyfikuje katabolizm glikokoniugatów w gruczołach ślino-wych, co można tłumaczyć zmianami czynnościowymi i morfologicznymi zachodzącymi w śliniankach. Znaczące zwiększenie wydzielania egzoglikozydaz w ślinie pacjentów z cukrzycą obu typów wskazuje na konieczność poświę-cenia szczególnej uwagi na zdrowie jamy ustnej tej grupy pacjentów (Adv Clin Exp Med 2013, 22, 5, 659–666).
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 hyperten-sion; 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 diagnos-tic dental tools (dental mirror and probe) follow-ing WHO criteria [13]. The dental status in all par-ticipants was determined by DMFT index (Decay, Missing, Filled Teeth), gingival status was exam-ined 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
collec-tion 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 sec-ond control (C II) – mean age 54 ± 3 years; DM-FT 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;
DM-FT 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 sali-vary 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 fluctua-tions related to a circadian rhythm of salivary se-cretion and composition. During saliva collection
the patient was seated on a chair and protected from any stimulation. The whole saliva was col-lected 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
min-utes at 4oC to remove cells and debris. The
result-ing 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 Mar-ciniak et al. [8]. HEX A specific activity was calcu-lated as the difference between the total HEX and HEX B activity.
Spectrophotometric measurements of p-ni-trophenol released by the exoglycosidases activity was carried out at 405 nm using microplate
read-er Elx800TM. Protein content in saliva was
deter-mined in duplicate by Lowry method [14].
Statistics
Statistical analyses were done using Statistica 10.0 (StatSoft, Cracov, Poland) according to ANO-VA and post hoc test. Pearson’s correlation coef-ficient was used to determine the association be-tween two variables. Results were expressed as means ± SD. Statistical significance was defined as p < 0.05.
Results
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 sali-vary HEX A in DM II group (616.49 ±528.66 pKat/ /kg of protein) was 2.9 times higher in compari-son 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 com-parison 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 compar-ison 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 significant-ly 1.3 times higher in comparison to the appropri-ate 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
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-hekso-zoaminidazy, HEX B – izoenzym B N-acetylo-β-heksoN-acetylo-β-hekso-zoaminidazy, 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,
of protein). The specific activity of salivary GLU in DM II group (171.07 ± 76.68 pKat/kg of pro-tein) was significantly 3.1 times higher in com-parison to the appropriate control (55.16 ± 15.09 pKat/kg of protein) (Fig. 1D). The output of sal-ivary 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 com-parison 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, postprandi-al glycaemia, glycaemia during sample collection,
HbA1C and the specific activities of
exoglycosidas-es examined.
Disscussion
The present study for the first time demon-strates that the salivary glands glycoconjugates and salivary glycoproteins catabolism, measured by the specific activity and the output of the lysosom-al exoglycosidases secreted into slysosom-aliva, 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 dia-betes, the significantly increased activity of salivary
Fig. 2. The output of salivary lysosomal exoglycosidases in DM I and II group in comparison to the appropriate con-trol. 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-signifi-cant increase.
total HEX is mainly due to the significantly in-creased HEX A specific activity and in less extent due to the moderate tendency to increase in HEX B specific activity. Significantly increased HEX spe-cific activity in DM II is an outcome of significant-ly increased HEX A as well as HEX B specific ac-tivities 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 re-sponsible for increasing total HEX and its isoen-zymes specific activity outside the cell- in saliva is not explained. The permeabilisation of lysosomal membrane and leakage of the enzymes via cyto-sol to the saliva by increased basement membrane permeability and/or cell membrane damage, hanced synthesis or delayed removal of these en-zymes 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 submandibu-lar 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 hypoth-esize 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 specif-ic 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 in-clude 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 di-abetes 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 neu-trophils infiltration and a release of primary gran-ules by neutrophils [17, 18].
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 function-al changes in the cells equfunction-ally influence the sfunction-ali- sali-vary 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 ac-tivity 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 au-thors. 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 ten-dency 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 solu-ble salivary glycoproteins (19) as well as epithe-lial cells components [20] containing numerous cell-membrane glycoproteins. Salivany glycopro-teins are an important part of oral defense: they take part in bacterial clearance and regulate bac-terial 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 high-ly effective lubricating properties [25] and main-tain pH of saliva [26]. One of the important parts in the degradation of oral glycoconjugates is de-glycosylation [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 in-creased similarly in both groups (Fig. 2). Signifi-cantly more exoglycosidases in diabetic patients saliva may accelerate the degradation of salivary and pellicle glycoproteins as well as glycosamino-glycans of oral mucosa surfaces and result in oral mucosa diseases, candidiasis, periodontitis, xe-rostomia and caries described by others in pro-fessional literature [28, 29].
saliva of both type diabetes patients once more in-dicates that special attention should be paid to the oral health of these patients. Therefore, diabetic
patients require special attention of diabetologists and special care of stomatologists.
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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
