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CLINICAL STUDIES |
1 Metabolic Bone Disease Unit, Rambam Medical Center, 31096 Haifa, Israel2 Biochemistry Laboratory and Salivary Clinic, Rambam Medical Center, 31096 Haifa, Israel3 Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, 31096 Haifa, Israel
(Correspondence should be addressed to R M Nagler who is now at Oral and Maxillofacial Surgery Department and Oral Biochemistry Laboratory, Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Rambam Medical Center, 31096 Haifa, Israel; Email: nagler{at}tx.technion.ac.il)
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Abstract |
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Methods: Out of 40 consenting female post-thyroidectomy patients, 23 (mean age 50±4 years old) were treated with I131 while 17 (mean age 46±4) were not. Whole saliva from all subjects was analyzed for antioxidant and biochemical composition and flow rate.
Results: The salivary flow rates of both groups were similar but their composition differed considerably. Salivary superoxide dismutase enzyme (SOD), total protein, and albumin concentrations were significantly reduced in the treated patients by 40, 25, and 18% respectively (P<0.05), as were all other salivary antioxidants. Oral discomfort complaints were far more prevalent in the I131-treated patients.
Conclusions: I131-dependent damage to the salivary glands was evidenced by a broad spectrum of compositional alterations and oral complaints. Reduction in salivary antioxidant status, SOD enzyme, and the uric acid molecule leaves the oral cavity less protected against oxidative stress. This is the first report of radioactive I131 treatment being harmful to salivary glands due to compromised salivary compositional and oxidative profile and oral discomfort complaints.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The purpose of the current study was to analyze the salivary flow rate and composition, including salivary antioxidants in radioactive I131-treated patients, and to concomitantly monitor salivary-related complaints.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In the current study, 40 consenting female patients, who had undergone surgery for thyroid carcinoma (either a total or a sub-total thyroidectomy) 8.4±7 years prior to the study, were enrolled. The post-surgical iodine absorption was 1±0.018%. Out of these 40 patients, 23 (mean age 50±4 years) were treated with I131 post-operatively (study group), while the other 17 (mean age 46±4 years) did not receive I131 at all (control group). The difference in the age of both groups was not significant. None of the patients smoke and they used general medications as required which are typical to their age group (such as for hypertension) but not any medication that is known to affect saliva. There was no significant difference in the percentage of the post-menopausal individuals in both groups (4 out of 17 of the non-iodine-treated group and 5 out of 23 of the iodine-treated group). There were no differences with respect to their TSH levels and only one post-menopausal patient of each group used hormone replacement therapy. The treated patients received I131 therapy at least 1 year prior to the study: 20 women received 100 mC, 2 women 150 mC, and 1 obese patient with neck lymph node metastasis received 200 mC.
In all subjects, salivary flow rates were measured, and saliva samples were studied for their biochemical and antioxidant composition. Concurrently, the patients were questioned about possible accompanying oral complaints. The biochemical analysis included various parameters reflecting the normal composition of human saliva (3): calcium (Ca), inorganic phosphate (P), magnesium (Mg), total protein (TP), albumin (Alb), lactate dehydrogenase (LDH), and amylase (Amy). The salivary oxidative analysis included the main salivary antioxidant molecule, such as uric acid (UA), the superoxide dismutase (SOD) enzyme, the peroxidase enzyme, and the total antioxidant status (TAS). The oral complaints monitored included mouth dryness, impulse to frequently wash/rinse the mouth, difficulty in swallowing, and changes in taste. All of these are known to be related to salivary alterations and are based on an established questionnaire previously used (4, 5).
Sialometrical analysis
Saliva was collected using the widely accepted procedure previously described (3), controlled to avoid drooling or swallowing. Whole saliva was collected in resting conditions in a quiet room during the morning, between 0900 and 1200 h, at least 1 h after food intake. Subjects were asked to collect saliva in their mouths and to spit it into a wide-mouthed test tube for 5 min. Saliva flow rates were estimated by measuring the volume of saliva thus collected, which was then centrifuged and kept at 4 °C until analyzed within 1 week of collection. Previous studies have shown that saliva remains stable during this time period with respect to the parameters analyzed in such conditions.
Salivary biochemical analysis
The P concentration was measured spectophotometrically, and Ca and Mg concentrations were measured by atomic absorption (6, 7). TP was measured as previously described (3) while amylase was measured by the Phadebas amylase test (Pharmacia Diagnostics). Albumin was measured by the radial immunodiffusion method (8), using an Oxford viewer for measuring the diameters of the precipitation rings (Mancini plates purchased from Binding Side, Birmingham, UK.) The diameter of the ring formed is quantitatively related to the concentration of various parameters analyzed. The LDH was measured at 37 °C by an optimized standard method using pyruvate as the substrate with the Hitachi 911 automated clinical chemistry analyzer using reagents purchased from Roche Diagnostics. The assay coefficient of variation (CV) was 2.1%. Amylase was measured at 37 °C using 4,6-ethylidene (G7)-p-nitrophenyl (G1)-
,D-maltoheptaoside as substrate, as previously described (9, 10); the assay CV was 3.4%.
Salivary oxidative analysis
Uric acid (UA) UA concentration was measured in the saliva of the patients using a kit supplied by Sentinel CH (Milan, Italy), as previously described (3). UA is transformed by uricase into allantoin and hydrogen peroxide that, under the catalytic influence of peroxidase, oxidizes the chromogen (4-aminophenazone/N-ethyl-methylanilin propan-sulfonate sodic) to form a red compound whose intensity of color is proportional to the amount of UA present in the sample and is read at a wavelength of 546 nm.
Peroxidase activity Peroxidase activity was measured in the patients' saliva according to the NBS assay, as previously described (11). Briefly, the calorimetric change induced by the reaction between the enzyme and the substrate, dithiobis-2-nitrobenzoic acid in the presence of mercaptoethanol, was read at a wavelength of 412 nm for 20 s.
Superoxide dismutase (SOD) activity The total salivary activity of the SOD enzyme (Cu/Zn- and Mn-SOD) was measured using the method of xanthine oxidase/XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt). The method is a modification of the NBT assay, in which XTT is reduced by superoxide radical generated by xanthine oxidase. Formazan is read at 470 nm. The SOD enzyme inhibits this reaction by scavenging superoxide radical. One unit of the enzyme is defined as the amount needed for a 50% inhibition of the absorption (12).
Total antioxidant status (TAS) TAS was assessed in the saliva samples, as previously described (3). Briefly, this assay is based on a commercial kit supplied by Randox (USA) in which metmyoglobin in the presence of iron is turned into ferrylmyoglobin. Incubation of the latter with the Randox (County Antrim, United Kingdom) reagent ABTS results in the formation of a radical colored blue-green, which can be detected at 600 nm.
Statistical analysis
For categorical variables, frequencies, percentages, and distribution were calculated. Distribution for categorical variables with small numbers of observations (at least one cell with less than five observations) was compared by Fisher–Irwin exact test. For continuous variables, ranges, medians, means, S.D., and S.E.M. were calculated. The results of age and time were compared by two-sample t-test for differences in means. Due to the large in-born variability of parameters in saliva, median values were calculated and compared by Wilcoxon rank-sum test (pairs of subgroups).
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Results |
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The salivary flow rate of both control and I131-treated groups was similar (0.29±0.04 and 0.32±0.04 ml/min respectively). However, the salivary composition of both groups was different. The median salivary Ca, P, and Mg concentrations in the control group were 2.7, 12.3, and 1.2 mg/dl respectively. In the I131-treated group, these values were lower by 26% (P=0.07), 14% (P=0.14), and 9% (P=0.15) respectively. The median salivary total protein and albumin concentrations were 70 and 59.4 mg/dl respectively, while the median enzyme activity of salivary LDH and amylase were 17 and 792 IU/l respectively. All four were lower in the saliva of the I131-treated group by 25% (P=0.007), 18% (P=0.05), 30% (P=0.45), and 31% (P=0.45) respectively (Table 1).
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The median salivary enzyme activity of the SOD in the control group was 0.61 U/ml, and in the I131-treated group it was 0.37 U/ml, a significant reduction of 40% (P=0.009). The median salivary TAS concentration of the control group was 0.47 mmol/l, and in the I131-treated group it was lower by 22% (P=0.34). The median UA concentration and peroxidase activity were lower by 5% in the I131-treated group, though these reductions did not reach statistical significance (Table 2).
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The rate of complaints of dry mouth was higher in the I131-treated patients when compared with the controls (33% vs 18%). Five of the treated patients complained of difficulty in swallowing and three complained of taste disturbances, while in the non-treated patients there were no such complaints. Thus, the complaint of difficulty in swallowing was found to be significantly more prevalent in the I131-treated group (P=0.05; Table 3). No significant correlations were found between any of the salivary changes and any of the subjective symptoms reported.
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Discussion |
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The I131-dependent damage, manifested by a broad spectrum of compositional alterations, may be of clinical merit especially as it is accompanied by various oral complaints. Moreover, the reduction in salivary TAS, the SOD and peroxidase enzymes, and the UA molecule is not only novel but it is also of significant clinical merit as the oral cavity and gastrointestinal tract remain less protected against oxidative stress. Until now, administration of post-thyroidectomy I131 has never been considered harmful to the salivary glands with respect to the salivary capacity to protect the oral cavity and the gastrointestinal tract against oxidative stress. The observation of the wide spectrum salivary compositional alteration unaccompanied by a salivary flow rate alteration indicates a specific salivary parenchyma insult. Otherwise, a diluting or concentrating effect due to salivary flow rate changes might have been postulated. Many of the salivary proteins and enzymes have functional and protective roles, and when their level is reduced, as in the current study, these roles are compromised. This phenomenon may have an accumulating, long-term effect on oral cavity health and possibly on the gastrointestinal tract into which the saliva is continuously swallowed after being secreted. The current observation of the significant reduction in salivary TP is of special importance, as this points at a wide spectrum reduction of various salivary enzymes and peptides rather than only a limited and specific one. Accordingly, one may conclude that many salivary functions are compromised. The reduction in the SOD activity by no less than 40% (P=0.009) is of special merit, as this enzyme is a pivotal one in the salivary antioxidant system and more so in the parotid gland that is known to be mostly sensitive to irradiation damage (12, 14). Moreover, the fact that not all decreases in the salivary parameters were statistically significant may be explained by the relatively small number of individuals analyzed, the high variability known to characterize saliva composition, and the relatively marginal effect of the radioactive I131 (15). Nevertheless, even if relatively marginal, the decreases in the salivary parameters may well have a long-term widespread debilitating effect on the saliva and its surroundings.
Regardless of the specific mechanism by which I131 affects the salivary glands, the data presented indicate damage to the glands induced by the therapy. Accordingly, administration of supportive therapy should be considered, based on agents including antioxidants, anti-inflammatory drugs, and saliva substitutes to the oral cavity of treated patients.
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Acknowledgements |
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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