Analysis of salivary parameters of mucopolysaccharidosis individuals

Abstract

Mucopolysaccharidosis (MPS) is a heterogeneous group of rare, chronic, progressive and systemic inherited disorders resulting from deficiency or lack of lysosomal enzymes responsible for the degradation of glycosaminoglycans. Products of nitrosative stress have been previously detected in blood and urine samples of patients with MPS. However, it is unclear whether they are present in the saliva of MPS patients and also if they correlate with salivary parameters such as flow and pH. This study compared the salivary levels of NO_X (NO₂^- + NO₃^-), nitrite (NO₂^-), protein (albumin), erythrocyte and leukocyte numbers, as well as the salivary flow rate and pH values of samples obtained from 10 MPS patients and 10 healthy subjects. MPS patients exhibited higher salivary levels of NO_X and NO₂^- when compared to healthy subjects (p < 0.05). Albumin was only detected in six saliva samples of MPS patients and, erythrocytes and leukocytes were detected in 60% and 40% of the MPS patients, respectively. In addition, salivary flow rate and pH averages were statistically lower in this group when compared to healthy samples (p < 0.05). Overall, the data indicates that the salivary levels of NO products can be used in combination with other heath indicators to monitor MPS disorders.

Mucopolysaccharidoses; Saliva; Oxidative Stress

Introduction

Mucopolysaccharidosis (MPS) is a heterogeneous group of rare, chronic, progressive and systemic inherited disorders resulting from deficiency or lack of lysosomal enzymes responsible for the degradation of glycosaminoglycans (GAGs). In MPS, GAG oligosaccharide accumulation occurs within the lysosomes of cells, inducing cell and tissue damage and dysfunction.¹ MPS is classified into seven groups (I, II, III, IV, VI, VII and IX) according with the type of enzyme deficiency involved in the disorder. With the exception of type II MPS, which is associated with the X chromosome, all other MPS consist in autosomal recessive disorders.²

The clinical characteristics of MPS vary with its type and, the heterogeneity of these signs is considerable.² Despite the correlation between some genotypes and phenotypes, individuals with identical enzyme deficiency may have distinct clinical magnitude (from mild to severe).³ The most commonly observed signs of MPS include umbilical and inguinal hernia, macrocephaly, short stature, marked skeletal alterations, corneal opacity, claw hands, facial dimorphism, macroglossia, included teeth, gingival hyperplasia and dental enamel defects.⁴ Diagnosis is based on traditional clinical and laboratorial criteria based on quantitative/qualitative urinary GAG analyses (e.g. dermatan sulphate; heparan sulphate; keratan sulphate and creatine) and enzyme activity assays (e.g. α-L-iduronidase, iduronate sulphatase and β-galactosidase). In addition, when available, molecular studies are important tools to identify MPS type.⁵

MPS types that prevalence at birth can vary among developed, underdeveloped and emerging countries.² In Norway, more than 60% of the MPS cases fall into the type I group.⁶ In contrast, data from Taiwan indicate that only 6% of the Taiwanese cases of MPS are classified as type I, and more than 50% as type II.⁷ In Brazil, it is estimated that 37% of the MPS individuals are type II. Conversely, in the United States, 31.7% of the MPS cases correspond to type IV. Thus, there is great inconsistency in the presentation of the disorder throughout the world.²

Saliva is a hypotonic fluid composed of elements produced by the salivary glands and constituents from blood plasma, such as hormones and cytokines, among others.⁸ Although saliva is not an filtrate of the blood plasma, changes in the systemic levels of substances such as cortisol, can be detected in it, indicating that this fluid could be potentially used as a source of disease markers s for diagnostic purposes.⁹ , ¹⁰ The use of saliva to monitor health and disease parameters is attractive as its collection is a non-invasive, easy and painless method that does not require specially trained individuals to collect samples,¹¹ opening new perspectives for diagnosis, early detection and monitoring of oral and systemic diseases.¹²

Changes in the redox status may occur in lysosomal storage diseases (LSD) such as MPS. Although the pathogenic mechanisms of LSD have been widely studied, they are not yet fully understood. In this context, reactive oxygen species (ROS) and nitrogen reactive intermediates (NRIs) may lead to increased lipid peroxidation of the lysosomal membrane and extravasation of its enzymes into the cytoplasm,¹³ therefore contributing to MPS. Inflammatory cell activation culminates in the up-regulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, an important enzymatic protein complex that reduces O₂ into reactive metabolites such as superoxide (O₂^-).¹⁴ O₂^-is an important substrate for the formation of hydrogen peroxide (H₂O₂) and peroxynitrite, the latter resulting from the reaction of O₂^- with nitric oxide (NO). Of note, peroxynitrite, can structurally alter cellular proteins, leading to cell death.¹⁵ Interestingly, blood and urine samples of type IV MPS individuals presented with an increased activity of superoxide dismutase, an enzyme responsible for the conversion of O₂^-into H₂O_2.¹⁵ Similarly, the RNIs have also been identified in blood and urine samples of MPS individuals.¹³ , ¹⁴ However, their salivary levels in MPS patients are yet to be established, as well as their correlation with other health salivary parameters. This study investigates the amounts of (NO₂^- + NO₃^-), nitrite (NO₂^-), protein (albumin), erythrocytes, leukocytes, pH and flow rate in saliva samples from MPS patients and healthy subjects. Possible correlations between these salivary parameters and the clinical signs of MPS such asvisible plaque, gingival bleeding on probing , caries and enamel defects were also determined.

Methodology

Ethical criteria and protocol

A clinical study with twenty individuals aged 5–22 years old was conducted after approval by the Ethics Committee of the Universidade CEUMA (UNICEUMA; Protocol Number: 1.756.478). The Informed Consent and Assent forms were signed by the guardians and/or participants. The protocol of this study followed the recommendations of the 2010 CONSORT statement (http://www.consort-statement.org).

Study design and participants

This cross-sectional study divided individuals into two groups (control and MPS). The control group consisted of 10 systemically healthy individuals with good oral hygiene who attended the dental clinic of UNICEUMA, in São Luís, Maranhão, Brazil. The MPS group was composed of 10 individuals with biochemical or molecular diagnosis of mucopolysaccharidosis regularly treated at the Hospital Materno Infantil, (São Luís, Maranhão, Brazil).

Individuals who made use of antibiotics or anti-inflammatory drugs in the month prior to the saliva collection were excluded from the study. For the control group, individuals with active carious lesions or periodontal disease were also excluded. A pilot study was carried out to evaluate the methodology and to calibrate examiners/evaluators. Individuals who participated in the pilot study were not included in the main study

Procedures

A questionnaire was applied to all participants for collection of demographic, socioeconomic and general health information. A detailed clinical examination was performed to assess the presence of dental caries,¹⁶ periodontal disease¹⁷ and enamel defects.¹⁶ The participants were asked not to eat two hours prior to the saliva collection. Saliva samples were acquired following dental prophylaxis with additive-free prophylactic paste,¹⁸ and were collected between 9–10 AM in order to minimize possible effects of the circadian rhythm.

Stimulated saliva samples were collected following chewing of plastic film (Parafilm®, 6.25 cm², Neenah, United States). The saliva collected within the first minute was discarded. Samples were then, deposited in sterile tubes kept on ice at every minute for 10 min. At the end of the last collection, the participants were instructed to expel the samples with the plastic film. Salivary flow was calculated as total saliva volume normalized by the collection time.¹⁸

All samples were centrifuged at 4°C, at 4000 rpm for 20 minutes, to remove debris and microorganisms present in the saliva. After centrifugation, the supernatant was collected and stored at -80°C, for further analysis.

NO₂^ˉ/NO₃^ˉ content was measured by the Griess colorimetric reaction, an indicator NO production. For this, NO₃^ˉ was reduced to NO₂^ˉ by incubating 80 µL of sample with 20 µL of 1U / ml nitrate reductase and 10 µL of 1 mM NADPH for 30 min, at 37°C, in a 96- well plate. Then, 100 µl Griess reagent, a 1: 1 mixture of 1% sulfanylamide and 0.1% of 5% naphthyl-enediamine-bi-chlorohydrate in orthophosphoric acid (H₃PO₄) were added per well and incubated for 15 min, at 37°C. The absorbance of each sample was determined at 550 nm in a spectrophotometer (MB-580/Heales, Shenzhen, China) and compared to a standard nitrite curve. Results were expressed as µM.

Additionally, the presence of NO₂^ˉ, albumin, erythrocytes, leukocytes and, pH values in saliva samples were measured by using reagent strips (Biocon 10, VerconDiagnostic, Belo Horizonte, Brazil). For this, each reagent spot of the strips was covered with 20 µl of saliva and incubated for 120 or 60 seconds, for leukocyte and other markers, respectively. After this period, a semi-quantitative reading was performed by visually comparing the color chart and the color intensities reflected by each area. For evaluation of NO₂^ˉ levels, a score (ranging from 1 to 3) was assigned, in which: 1=absence of NO₂^ˉ, 2 = small/moderate amount of NO₂^ˉ and 3 = large amount of NO₂^ˉ. Protein (albumin) quantification results were also presented in scores (ranging from 1 to 3), where: 1=presence of albumin, 2 = moderate amounts of albumin and 3 = large amounts of albumin. For the evaluation of erythrocytes and leukocytes, the score ranged from 1 to 4 where: 1= absence, 2=traces, 3=moderate and 4= large amounts of white or red cells. The pH evaluation (5 to 9) was assessed by visually performing a comparison between the color chart and the color intensities of the strips after the addition of saliva.

Data analysis

Data are represented as mean ± SD or median and interquartile range (25–75^th; IQR), depending on their distribution. Accordingly, we used parametric (unpaired t-test) or non-parametric (Mann-Whitney) tests to determine the significance of differences between groups in the NO_x values and NO₂, albumin, pH, erythrocytes and leucocytes scores. Correlations between the different parameters were determined using Spearman’s rho. Socio-demographic and oral health data on the number of decayed and permanent teeth were compared between groups by Fisher’s exact test. The medians of continuous variables such as visible plaque index, gingival bleeding on probing index and salivary flow were compared by Wilcoxon ranking tests. Correlations between the evaluated parameters were measured by Spearman’s correlation test. Significance level of 5% was considered for all analyses. Analyses were performed using Stata 16.0 software (StataCorp LLC, CollegeStation, USA).

Results

According to Table 1 , MPS and control groups were composed of 60% of individuals aged 5 to 12 years old and 40% of individuals aged 13 to 22 years old. Both groups showed similar gender distribution, and most MPS individuals identified themselves as African Americans (70%). When inquired about their oral health, 70% of the individuals on the MPS group and 100% the individuals on the control group rated themselves as excellent and 30% of the patients from the MPS had a poor oral health. MPS patients and control groups were comparable in regards to numbers of decayed permanent and deciduous teeth ( Table 1 ), the data shows that 100% of the patients of the control group did not have any type of decayed teeth, however for the MPS group 33% of patients presented at least one or more permanent/deciduous decayed teeth. Median and IIQR values observed for visible plaque index was higher in the MPS group compared with the control group (52.7 (17.5–97.9) versus 0; p< 0.001). The median of the gingival bleeding index was also higher in the MPS group in comparison with the control group (16.2 (0.0–45.5); versus 0; p = 0.022). Salivary flow rate was significantly higher (p = 0.007) in healthy subjects when compared to MPS, 0.8 and 0.3 mL/min, respectively ( Table 1 ). Median and IQR values observed for albumin present in saliva are as follows: healthy subjects 1.0 (1.0–1.0) and MPS 2.0 (1.0–2.0) with a statistical difference between groups, p = 0.0087 ( Figure A ). In addition, the pH median and IQR values for healthy subjects and MPS where 7.0 (7.0–7.5) and 7.0 (6.0–7.0) being statistically different, p = 0.0074 ( Figure B ).

Table 1
Patients characteristics.

Characteristics	Healthy subjects	MPS	p-value*
	n (%)	n (%)
Agea (years)			1.000
5–12	6 (60%)	6 (60%)
13–22	4 (40%)	4 (40%)
Gendera			1.000
Male	5 (50%)	5 (50%)
Female	5 (50%)	5 (50%)
Skin colora			0.350
White	5 (50%)	2 (20%)
Brown	5 (50%)	7 (70%)
Black	0 (0%)	1 (10%)
Oral healtha			0.211
Good	10 (100%)	7 (70%)
Poor	0 (0%)	3 (30%)
Decayed permanent teetha			0.087
0	10 (100%)	6 (67%)
1 +	0 (0%)	3 (33%)
Decayed deciduous teetha			0.417
0	3 (100%)	4 (67%)
1 +	0 (0%)	2 (33%)
	Median (IQ)	Median (IQ)
Visible plaque index (%)b	0.0 (0.0–0.0)	52.7 (17.5–97.9)	< 0.001
Gingival bleeding index (%)b	0.0 (0.0– 0.0)	16.2 (0.0–45.5)	0.022
Salivary flow rate (mL/min)b	0.8 (0.6–1.5)	0.3 (0.2–0.3)	0.007

• IQ: Interquartile Range; ^aFisher’s exact test; ^bWilcoxon rank test.

Figure
Median and IQR values observed for albumin present in saliva (Figure A). Salivary pH median and IQR values for healthy subjects and MPS (Figure B). Amounts of nitrite observed in healthy and MPS patients (Figure C). Salivary NOx values (Figure D). Erythrocytes (Figure E) and Leukocytes (Figure F) scores in the saliva.

Higher amounts of nitrite were observed in MPS patients, 3.0 (2.75–3.0), when compared to the control group, 2.0 (2.0–2.0), p = 0.0105 ( Figure C ). Figure D , presents the mean and standard deviations of NO^x in saliva, showing that patients with MPS had a higher amount (p=0.0386) of nitrite and nitrate (111.6 ± 51.8) in their saliva when compared to the healthy subjects (69.8 ± 28.5).

Erythrocytes were detected in 30% of the individuals of the control group in contrast with 90% of MPS patients ( Figure E ). I In both groups, three individuals presented traces of hemoglobin in their saliva, with values ranging from 0.1-10 RBC/µL of sample, in addition, six patients from the MPS presented a moderate or large amount of hemoglobin in their saliva with a variation of 50 to 250 RBC/µL.

Leukocytes were detected in the saliva of 10% of the healthy individuals and in 90% of the MPS patients. Among the MPS patients, six presented traces, two moderate amounts and one, a large amount of leukocytes in their saliva. In addition, the three MPS individuals with the highest amounts of leucocytes also presented the highest levels of salivary NO_x ( Figure F ).

Gingival bleeding on probing and visible plaque indexes were positively correlated with protein in the MPS group (r = 0.909 and r = 0.898, respectively). In addition, positive correlations were observed between the number of decayed teeth and protein (r = 0.877), and also, between the number of teeth with enamel defects (r = 0.800) and protein levels in MPS individuals ( Table 2 ).

Table 2
Spearman’s correlations coefficient between the Clinical/Biochemical parameters and NOX levels in saliva (n = 20).

Variables	Healthy subjects			MPS
	NOx	pH	Protein	NOx	pH	Protein
Visible plaque index	-	-	-	0.109	-0.233	0.898**
Gingival bleeding index	0.174	-0.266	-	-0.025	-0.007	0.909**
Salivary flow rate	-0.337	0.385	-	-0.559	0.219	-0.436
Number of decayed teeth	-	-	-	0.082	-0.125	0.877**
Number of teeth with defects	-	-	-	-0.225	-0.187	0.800*

• *p < 0.05; **p < 0.001

Discussion

Saliva has been used as a tool for the diagnosis, early detection, progression and monitoring of various systemic diseases.⁸ , ⁹ Although the pathogenic mechanisms of MPS have been widely studied, they are not fully understood. Thus, the evaluation of nitrosative stress parameters and other biomarkers in saliva of MPS individuals may help understanding the underlying causes of the disease. According to our results, the salivary parameters evaluated in this clinical study can be considered potential biomarkers for disease monitoring.

The evaluation of NO_x levels is an indirect way to measure nitrite and nitrate levels in saliva.¹⁹ To date, this is the first study to quantify salivary nitric oxide and nitrite levels in MPS patients. Based our data, it was found that the MPS group presented higher NO^x production and nitrite in saliva samples when compared to the control group (p = 0.00386). Blood and urine samples analysis from patients with type II MPS undergoing long-term Enzyme Replacement Therapy (ERT), showed a positive correlation between GAG levels and urinary nitrate and nitrite concentrations, as well as between IL-1β and NO^x plasma concentrations, presenting increased plasma and urine ROS/NRIs. These findings reinforce that, even under ERT, a NO-dependent inflammation is present in this serious disease.¹³

Interestingly, individuals with type II MPS under ERT exhibited an increase in NO and also in IL-1β.¹³ Individuals with type II MPS under ERT showed increased NO, NO₂^● and NO₃⁻ + NO₂⁻, but not superoxide dismutase activity or ONOO- levels ¹³ . NLRC4 inflammassome has been associated with pathogenic microorganisms, while NLRP3 has been associated with sterile inflammation; however, recent studies²⁰ , ²¹ have shown that NLRC4 can be activated in the absence of microorganisms by molecular patterns associated with damage.

Studies have indicated that GAG accumulation would be responsible for triggering inflammation and that even in individuals under ERT, there is an increase in proinflammatory mediators.¹³ , ²² , ²³ , ²⁴ Thus, we believe in the potential involvement of NO in the induction and maintenance of inflammatory states in MPS individuals.

NO is considered one of the salivary components with promising potential for prediction of inflammatory events and its role in the pathogenesis of oral diseases has been investigated ² , ²⁵ . A recent study associated total salivary nitrate and nitrite levels with periodontal health in 74 individuals.²⁵ The authors observed that the increase in salivary nitrate and nitrate concentrations was related to periodontal changes and that periodontal treatment led to a decrease in the levels of these markers. The same study concluded that salivary nitrate and nitrite levels are associated with defense mechanisms and suggested that salivary glands may respond to infectious oral diseases, increasing the release of NO.²⁵

In the present study, positive correlations were observed between protein and gingival bleeding on probing, visible plaque indexes, number of decayed teeth and number of teeth with defects in MPS patients, with a significant statistical difference vs control. When we correlated the same parameters with only the dosed nitrite in reagent strips, a weak correlation was obtained. It is known that there is an increase in the production of oral nitric oxide during the beginning of dental plaque deposition.²⁶ Similar studies suggest that this increase results from overexpression of the gingival cell-induced nitric oxide synthase (iNOS) enzyme. Such excess is not only derived from bacterial proliferation, but also from cytokine production stimulated by the presence of bacterial plaque.²⁷ , ²⁸ Overall, the protein increase is mostly involved in tissue protection and inflammatory control, suggesting the activation of protective pathways, including antibacterial activity, regulation of the inflammatory process, antioxidants, and protease inhibitors characterizing the host responses. The negative moderate correlation between salivary flow rate and NO_x in the MPS patients indicates that oxidative stress causes changes in the structure of the salivary gland and is involved in hyposalivation. The accumulation of GAGs in the cells causes an inflammatory response and it is mediated by neutrophils migrating from the blood stream to the gingival tissues and into the oral cavity through the GCF or saliva. We speculate that the increase in oral erythrocytes, leucocytes and NO_x in MPS patients is related to cell disruption caused by the GAG accumulation.

During the inflammatory response, NO production in the oral cavity related to periodontal disease occurs via iNOS. Fibroblasts, inflammatory infiltrate cells, and other periodontal cells begin to produce NO via iNOS. Despite being a defense mechanism, excessive amounts of this free radical may contribute to tissue destruction in pathogenic periodontitis.²³ , ²⁶ Considering that MPS individuals in this study presented plaque and gingival bleeding on probing indexes and high NO_x levels, added to plaque retentive factors, such as enamel defects, it is suggested that these individuals are more likely of developing periodontal diseases.

Some plasma-derived diagnostic markers, such as albumin, can be found in saliva. Albumin is the major plasma protein and its concentration in saliva depends on periodontal health, presence (or absence) of teeth and oral mucosa integrity.²⁹ Helmerhorst et al.²⁹ demonstrated that plasma-derived albumin concentration increased after oral hygiene ceased and returned to baseline levels after oral hygiene resumption in individuals with gingivitis. The present study evaluated the albumin levels in saliva from individuals in both groups. When the control group was compared to the MPS, it was possible to observe that MPS presented higher levels of albumin, where 70% of the patients presented albumin in a range of 30 to 100 mg/dL, according to Carda et al.³⁰ the usual amount of albumin in saliva should not be higher than 30 mg/dL. There was strong correlation between this plasma protein and gingival bleeding on probing index (r = 0.909, p < 0.001) and visible plaque index (r = 0.898, p < 0.05), confirming that of periodontal health influences the albumin concentration in saliva.

When the presence of erythrocytes in saliva is analyzed by the reagent strips, tree samples from the control group had red blood cells, even though clinically bleeding on probing was not detected, this could possibly be explained by trauma occurring during dental prophylaxis or even by chewing the plastic film. Interestingly, 90% of the patients of MPS group presented higher amounts of RBC in their saliva. In addition, individuals with type II MPS have elevated saliva leukocyte levels. This could be explained by the continued use of Risperidone, since this drug consists of an anti-psychotic that may present leukocytosis as adverse effect. Moreover, we discovered that the same individual was making use of unsuitable enzyme doses. Thus, it is possible that this may have decreased the expected protective effect of ERT and may have contributed to both the leukocytes increase and the higher NO^x levels. In contrast, this individual did not have a higher level of salivary albumin, despite the presence of leukocytosis, which makes it difficult to attribute high saliva leukocyte levels to local inflammatory factors such as periodontal disease. As this is an unusual finding, it is likely that such an occurrence is not of local inflammatory nature, such as gingivitis, but of a systemic nature, since local inflammatory response would cause the presence of albumin in saliva.

The average plaque index found in our results was 54.08%, and half of individuals had indexes greater than 50% and three of these indexes greater than 95%. These results support the hypothesis that MPS individuals are more likely of having caries and periodontal disease. Only three individuals had no gingival bleeding on probing Orofacial alterations observed in these individuals, such as malocclusion, mouth opening limitations, macroglossia and enamel defects,³¹ , ³² in addition to motor and intellectual limitations, may have contributed to the oral condition observed in this study.

A strong correlation between the number of teeth with enamel defects and the number of decayed teeth was also observed. The enamel of MPS individuals is fragile and more porous,³³ , ³⁴ , ³⁵ especially those with type IV MPS. These defects topographically modify the dental enamel, facilitating bacterial adhesion.³⁶ Some dental structural alterations have been described in type I PMS, such as narrow dentinal tubule, dentin deposition in irregular wave pattern, small cracks in the enamel-dentinal junction, altered arrangement of enamel prisms.³⁵ Deficiency of the N-acetyl galactosamine-6-sulfatase enzyme in IVA type MPS results in the pathological accumulation of GAG keratan sulfate and chondroitin 6-sulfate in the secretory phase ameloblast lysosomes, contributing to the formation of defective enamel.³⁷

Stimulated salivary flow < 0.8ml/min is considered a pathological condition and has been considered a strong indicator of increased caries risk.³⁷ , ³⁸ , ³⁹ In the present study, salivary flow rate and pH of stimulated saliva were reduced in MPS individuals when compared to controls, corroborating previous studies.³⁸ Eight subjects had salivary flow rate less than or equal to 0.3 ml / min. We believe that the causes of hyposalivation in this group of individuals are related to the orofacial characteristics that influence chewing performance⁴⁰ and the possible GAG accumulation in the salivary glands, since there is evidence of GAG accumulation in organs such as liver, kidneys and spleen in IIIC type MPS models.⁴⁰

In addition, it was possible to observe that MPS individuals presented lower salivary pH values when compared to the heathy group, which does not corroborate literature findings about the influence of salivary pH reduction on the dental caries process.³⁹ However, some studies have shown correlation between these two findings.³⁹ A positive correlation between salivary flow and pH was observed in our results, corroborating a study³⁸ in which MPS individuals presented lower salivary flow and pH averages compared to healthy controls.³⁸ Furthermore, the correlation between visible dental plaque index with salivary flow and pH was investigated, resulting in negative correlation among these parameters. These data reinforce the increased risk of caries in these individuals.

In conclusion, our data indicate that the salivary parameters evaluated in this study could help understanding the pathophysiology of mucopolysaccharidosis. Thus, we suggest the quantification of these salivary parameters to comprehensively monitor the disease stage and development.

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Publication Dates

• Publication in this collection
  24 Jan 2022
• Date of issue
  2022

History

• Received
  13 May 2021
• Accepted
  31 Aug 2021
• Reviewed
  16 Sept 2021