o t o l a r yn go l o g i a p o l s k a 6 7 ( 2 0 1 3 ) 2 0 4 – 2 0 8

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Original research article/Artykuł oryginalny

N-acetyl-b-hexosaminidase in chronic tonsillitis and tonsillarhypertrophy

Mariola Zagor 1,*, Alina Minarowska 2, Małgorzata Knaś 3, Katarzyna Krajewska 4,Anna Niemcunowicz-Janica 5, Justyna Marciniak 6, Marcin Bierć 7,Agnieszka Zaniewska 8, Łukasz Minarowski 9, Anna Jackowska 10,Tomasz Jackowski 10, Krzysztof Zwierz 8, Sławomir Szajda 11

1Department of Otolaryngology, Stomatology Division, I Faculty of the Warsaw Medical University, Warsaw, Poland2Department of Surgical Nursing, Medical University of Bialystok, Poland3Research Laboratory of Cosmetology, Medical University of Bialystok, Poland4Department of Otolaryngology Voivodship Hospital in Bialystok, Poland5Department of Forensic Medicine, Medical University of Bialystok, Poland6Department of Pharmaceutical Biochemistry, Medical University of Bialystok, Poland7Department of Oral Surgery, Medical University of Bialystok, Poland8Medical Institute, Medical College of the Universal Education Society, Lomza, Poland9Department of Lung Diseases and Tuberculosis, Medical University of Bialystok, Poland10Department of Cardiology, J. Śniadecki Regional Specialist Hospital, Bialystok, Poland11Department of Emergency Medicine and Disasters, Medical University of Bialystok, Poland

a r t i c l e i n f o

Article history:

Received: 13.05.2013

Accepted: 22.05.2013

Available online: 24.05.2013

Keywords:� N-acetyl-b-hexosaminidase (HEX)� Palatine tonsils� Chronic tonsillitis� Tonsillar hypertrophy

a b s t r a c t

Background: The concentration and specific activity of N-acetyl-b-hexosaminidase (HEX) in

palatine tonsils with chronic tonsillitis and tonsillar hypertrophy give insight in tonsillar

tissue remodeling and constitute a potential marker for diagnosis and treatment of chronic

tonsillitis and tonsillar hypertrophy. Aim: Determining the concentration and specific acti-

vity of N-acetyl-b-hexosaminidase in palatine tonsils with hypertrophy and chronic tonsil-

litis. Methods: HEX activity was analyzed by the method of Marciniak et al. with p-nitrop-

henyl N-acetyl-b-glucosaminepyranoside as a substrate. Results: The concentration and

specific activity of HEX in palatine tonsils in patients with tonsillar hypertrophy and chro-

nic tonsillitis both in childhood and adulthood significantly increase in comparison to

healthy individuals. Conclusions: Our data demonstrate the presence of HEX in palatine

tonsils and indicate on significant increase of its concentration and specific activity. Based

on content and specific HEX activity we suggest that tonsils with hypertrophy and chronic

tonsillitis should be treated as identical unit irrespectively of age.

© 2013 Polish Otorhinolaryngology - Head and Neck Surgery Society. Published by

Elsevier Urban & Partner Sp. z o.o. All rights reserved.

* Corresponding author at: Department of Otolaryngology, Stomatology Division, I Faculty of the Warsaw, Medical University Warsaw,Poland. Tel.: +48 694 012 224.

E-mail address: popkom@interia.pl (M. Zagor).0030-6657/$ – see front matter © 2013 Polish Otorhinolaryngology - Head and Neck Surgery Society. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

http://dx.doi.org/10.1016/j.otpol.2013.05.003

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Introduction

The palatine tonsils are the largest component of Waldeyertonsillar ring. The location of Waldeyer tonsillar ring and itsdesign allow direct exposure of the immunologically activecells to foreign antigens entering the upper aerodigestivetract, which maximizes the immunologic response [1]. Thetonsillar structures maximize the exposure of tissue tosurface antigen, harbor debris and bacteria which may bethe reason why tonsils are so commonly infected andhypertrophied. Chronic tonsillitis and tonsillar hypertrophyare often encountered diseases in otolaryngology, respon-sible for a significant proportion of childhood and adulthoodillnesses. Although the criteria for tonsillectomy or subtotal(i.e. intracapsular) tonsillectomy have been proposed,otolaryngologist still debate the duration of conservativetreatment and the necessity of performing the subtotaltonsillectomy [2, 3]. Therefore, there is a need for betterunderstanding of palatine tonsils diseases. We suggest thatone of such method may be the assessment of lysosomalexoglycosidases activity.

N-acetyl-b-hexosaminidase (HEX), the most active lyso-somal exoglycosidase, contributes to degradation ofa tonsillar oligosaccharide chains of glycoconjugates: glyco-proteins, glycolipids and proteoglycans [4]. The increase ofHEX activity was reported in patients with chronic inflam-matory joint, kidney disease and salivary gland tumors[5–7]. HEX activity has been proposed as biochemical markerof tissue injury, inflammation, malignancy and alcoholabuse [8–14]. Therefore, we decided to investigate contribu-tion of HEX activity in pathogenesis of chronic tonsillitisand tonsillar hypertrophy. The objectives of our study wereas follows: evaluation of HEX presence in healthy palatinetonsils (C), tonsillar hypertrophy (H), chronic tonsillitis inchildren (TC) and adults (TA); comparison the HEX activityamong C, H, TC, TA patients' subsets.

Materials and methods

Palatine tonsils

Palatine tonsils were obtained from patients who underwentsubtotal or total tonsillectomy due to tonsillar hypertrophy(H) (n = 15), chronic tonsillitis in children (TC) (n = 8) andchronic tonsillitis in adults (TA) (n = 22) in Department ofPediatric Otolaryngology, Medical University of Bialystok,and Department of Otolaryngology Voivodship Hospital inBialystok. Healthy palatine tonsils, serving as a controlgroup (C) (n = 15), were obtained in Department of ForensicMedicine, Medical University of Bialystok from young, heal-thy individuals, within 14 h after sudden decease. The studywas approved by the local ethics committee, and all patientsor parents of patients signed an informed consent form.

Tissue samples

The samples, removed from palatine tonsils, were washedwith 0.9% NaCl, weighed and homogenized at 4 8C in an

Ultra Turrax T8 homogenizer in 10 volumes of 0.15 M KClcontaining 0.2% Triton X-100. The resulting homogenateswere centrifuged at 10,000 � g for 30 min at 4 8C. Thesupernatants were used as crude enzyme solution.

Assay for exoglycosidase activity

The activity of HEX (EC 3.2.1.55) was determined by themethod of Marciniak et al. [15]. The substrate for HEX was6.67 mM solution of p-nitrophenyl-b-D-N-acetylglucosamine-pyranoside (Sigma, St Louis, MO, USA), in 0.1 M citrate-phosphate buffer at pH 4.7, stored at �20 8C. The substratewas incubated for 60 min at 37 8C with appropriately dilutedsupernatant with 0.1 M citrate-phosphate buffer. The reac-tion was stopped by adding 0.2 M borate buffer, pH 9.8.

The measurements of the released p-nitrophenol werecarried out at 405 nm using a spectrophotometer (Spekol 11,CARL ZEISS JENA). The concentrations of the HEX activitywere expressed as nanokatales (nanomoles of p-nitrophenolreleased per second) per mL of tonsillar supernatant andspecific activity as milikatals/kg of the supernatant proteinsfrom tonsillar tissue.

Statistical analysis was performed using Statistica 9.0(Statsoft, Cracov, Poland). Kruskal–Wallis ANOVA, Medianand LSD tests were used to study the significant differencesbetween groups. Statistical significance was defined asp � 0.05.

Results

The concentration of the HEX activity in tissue with tonsillarhypertrophy (124.3 nkat/mL), chronic tonsillitis both inchildhood (123.4 nkat/mL) and adulthood (128.7 nkat/mL)significantly increase in comparison to healthy palatinetonsils (78.1 nkat/mL). The concentration of the HEX activityis 1.5 times greater for both hypertrophied tonsils andchronic tonsillitis in children and 1.6 times greater forchronic tonsillitis in adults, in comparison to healthytonsillar tissue (Table I, Fig. 1).

The specific HEX activity accounts for 18.6 mkat/kg oftonsillar supernatant proteins in the hypertrophied tonsils,17.9 mkat/kg for chronic tonsillitis in children's tonsillartissue and 18.9 mkat/kg for chronic tonsillitis in adult'stonsillar tissue. The specific HEX activity of tonsillar super-natant proteins is 1.6; 1.5 and 1.6 times greater, respectivelyin comparison to healthy tonsillar tissue (Table II, Fig. 2).

Discussion

The immune activity of the tonsillar tissues is most promi-nent from the ages of 4 to 10 and tends to involute afterpuberty. After involution, the secretory immune function oftonsillar tissues remains, but at decreased level. Accordingto Paulussen et al. [16], due to important, but not completelyinvestigated immune function of the tonsillar tissues andsignificant postoperative morbidity it is essential to limitindications for tonsillectomy. This raises the question: isthere a difference in metabolism of tonsillar tissue in

Table I – The HEX activity (nkat/mL) in human palatine tonsils

N Medium (nkat/mL) SD Ratio H/C, TC/C, TA/C

C 15 78.1 7.0H 15 124.3 8.2 1.5TC 7 123.4 8.8 1.5TA 23 128.7 13.9 1.6

[(Fig._1)TD$FIG]

Fig. 1 – The HEX activity (nkat/mL) in human palatine tonsils

[(Fig._2)TD$FIG]

Fig. 2 – The specific HEX activity (mkat/kg protein) in humanpalatine tonsils

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chronic tonsillitis and tonsillar hypertrophy. In the presen-ted study, we have attempted to answer above question bydetermination of N-acetyl-b-hexosaminidase (HEX) that maycontribute to degradation of the tonsillar connective tissuecomponents, inter alia glycoconjugates, in chronic tonsillitisand tonsillar hypertrophy.

Our study demonstrated the presence of highly activeHEX in healthy and pathologic tonsillar tissue both inchildren and adults, which suggest that indeed, HEX intonsillar tissue is involved in degradation of oligosaccharidechains of glycoconjugates. The latter include glycoproteinsand glycolipids [17] constituting cellular membranes, as wellas heteropolysaccharide chains of glycosaminoglycans andproteoglycans constituting with glycoproteins extracellularmatrix. Oligosaccharide chains are released from glycocon-jugates by endoglycosidases (e.g. hyaluronidase or chondroi-tinases in the case of glycosaminoglycans) and then succes-sively cleaved at the non-reducing end of oligosaccharideschains by concerted action of exoglycosidases, and in thecase of HEX, resulting in release single N-acetylglucosamineor N-acetylgalactosamine units [18].

Table II – The specific HEX activity (mkat/kg protein) in human

N Medium (mkat/kg)

C 15 11.6H 15 18.6TC 7 17.9TA 23 18.9

Our results indicate that concentration and HEX specificactivity in hypertrophied tonsils and chronic tonsillitis inboth children and adults were significantly greater than inhealthy individuals, whereas there was only negligibledifference in HEX concentration and specific activity bet-ween both hypertrophied tonsils and individuals with chro-nic tonsillitis. Our results accord to those obtained by Zoch-Zwierz and Rudobielska [6] regarding chronic renal insuffi-ciency and Popko et al. [5] concerning rheumatoid arthritisand juvenile idiopathic arthritis.

On the basis of HEX activity, it can be concluded thattonsillar hypertrophy and chronic tonsillitis can be treatedas identical units. We assume therefore that although thesubtotal tonsillectomy may offer a decreased rate of posto-perative morbidity [19], in the long-standing outcome it cancontribute to transformation of preserved tonsillar tissueinto chronic tonsillitis.

Furthermore, we have observed that there is no signifi-cant difference in concentration and HEX specific activitybetween chronic tonsillitis in children and adults. Wehypothesize therefore that the tonsillar remodeling is

palatine tonsils

SD Ratio H/C, TC/C, TA/C

1.11.6 1.62.2 1.51.7 1.6

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equally intensive both in childhood and in adulthood. Thus,there is an increasing body of evidence disapproving theneed of subtotal tonsillectomy.

We have provided evidence that not only chronic tonsilli-tis but also tonsillar hypertrophy comparably accelerate thetonsillar tissue remodeling, reflected in increased HEXactivity, eventually leading to tonsillar tissue impairment.Theoretically, the removal of pathological tonsils shoulddecrease the HEX activity either in blood serum, saliva orurine. Therefore, determination the HEX concentration andspecific activity in body fluid before and after tonsillectomycould constitute a plausible alternative to HEX examinationin palatine tonsils in vivo. Further study on this hypothesisis needed, but it may offer additional details about specificfeatures, monitoring and treatment of particular tonsillardiseases. Our assumption agrees with a study of GarciaCallejo et al. [20] who examined activity of superoxidedismutase (SOD) in blood and palatine tonsils before andafter tonsillectomy. They concluded that SOD activity inpalatine tonsils and/or peripheral blood increases proportio-nally to infections incidence, which allows detectingpatients with functional tonsillar tissue damage, and objec-tively recommending tonsillectomy or monitoring clinicalresponse for a therapy.

As the determination the HEX activity is a simple, robust,non-invasive and inexpensive, we speculate that the HEXactivity in body fluids determined before and after tonsillec-tomy could be a potential marker for monitoring the courseof particular palatine tonsils' diseases.

Conclusions

In the present study, we have demonstrated the presence ofHEX in palatine tonsils and depicted its concentration andspecific activity differences between healthy individuals andthose with chronic tonsillitis and tonsillar hypertrophy. Weconcluded that the significant increase of HEX concentrationand specific activity in well-defined group subsets proveincreased glycoconiugates' catabolism contributing to tonsil-lar tissue damage. Our results established a sound basis fortreating tonsillar hypertrophy and chronic tonsillitis asidentical unit irrespectively of age. Furthermore, we suggestthat it would be of a particular interest to examine HEXconcentration and specific activity in body fluids before andafter tonsillectomy. It could constitute a potential markerfor diagnosis and treatment of chronic tonsillitis and tonsil-lar hypertrophy.

Authors' contributions/Wkład autorów

MZ was responsible for study design, data collection andinterpretation, statistical analysis, acceptance of finalmanuscript version, literature search, funds collection. AMwas responsible for study design, data collection and inter-pretation, acceptance of final manuscript version, literaturesearch. MK was responsible for study design, data collectionand interpretation, acceptance of final manuscript version. KKwas responsible for study design, data collection, acceptance

of final manuscript version, literature search. AN-J wasresponsible for data collection, statistical analysis, acceptanceof final manuscript version, literature search. JM, MB and AZwere responsible for data collection, statistical analysis,literature search. ŁM was responsible for statistical analysis,data interpretation literature search. AJ and TJ were respon-sible for statistical analysis literature search. KZ and SS wereresponsible for data interpretation, acceptance of finalmanuscript version, funds collection.

Financial support/Finansowanie

The study was financed from the grant of Medical Universityof Bialystok.

Conflict of interest/Konflikt interesu

None declared.

Ethics/Etyka

The work described in this article have been carried out inaccordance with The Code of Ethics of the World MedicalAssociation (Declaration of Helsinki) for experiments invol-ving humans; EU Directive 2010/63/EU for animal experi-ments; Uniform Requirements for manuscripts submitted toBiomedical journals.

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