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Destruction of Parotid Glands Affects Nitrate and Nitrite Metabolism

¹ Salivary Gland Disease Center, Faculty of Stomatology, Capital University of Medical Sciences, Tian Tan Xi Li, No. 4, Beijing 100050, PR China; and
² Peking University School of Oncology & Beijing Institute for Cancer Research, Beijing 100034, PR China;

Correspondence: *corresponding authors, songlinwang{at}dentist.org.cn

	ABSTRACT

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The role of salivary glands in nitrate and nitrite metabolism is poorly understood. The aim of the present study was to investigate the effect of parotid gland ablation on dynamic metabolism of nitrate and nitrite in miniature pigs. The parotid glands of 5 healthy miniature pigs were bilaterally ablated by methyl violet. Concentrations of nitrate and nitrite of whole saliva, serum, and urine samples were analyzed by high-performance liquid chromatography. Results showed that bilateral ablation of the parotid glands led to a significant decrease of nitrate secretion from blood to saliva (P < 0.05) and thus low nitrite levels. Dysfunction of the parotid glands temporarily increased the systemic level of nitrate in miniature pigs after nitrate loading. This study suggests that the parotid glands play an important role in the balance of nitrate and nitrite levels in both whole saliva and the body.

Key Words: saliva • parotid gland • nitrate • nitrite • miniature pig

	INTRODUCTION

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Nitrate is a natural component existing in many fruits and vegetables and in drinking water. In the oral cavity, dietary nitrate is partially converted into nitrite by nitrate reductase derived from commensal bacteria (Tannenbaum et al., 1976). Nitrite is a precursor of N-nitroso compounds, which may play a role in gastric/intestinal carcinogenesis (Hill et al., 1973; Airoldi et al., 1997; Deng, 2000). It reacts with amines and amides in stomach endogenously to form carcinogenic N-nitroso compounds (Mirvish, 1983; Schweinsberg and Burkle, 1985; Shapiro et al., 1991; Deng and Xin, 2000). In contrast, dietary nitrate is the basis for a non-immune defense mechanism against oral and intestinal pathogens in humans and animals (Duncan et al., 1995). Therefore, salivary nitrate/nitrite may contribute to the host defenses against ingested pathogens (Dougall et al., 1995).

Saliva collected directly from either parotid or submandibular salivary ducts contains nitrate but no nitrite, while mixed whole saliva contains both (Tannenbaum et al., 1974; Ishiwata et al., 1975a). To our knowledge, the kinetics of nitrate in saliva, serum, or urine after nitrate intake has been little studied (Cortas and Wakid, 1991). Hence, the purpose of this study was to describe the dynamic fluctuations of nitrate and nitrite levels in saliva, serum, and urine following an oral administration of KNO₃ in a group of parotid sialectomized miniature pigs to investigate the role of the parotid glands in nitrate and nitrite metabolism. Miniature pigs were used as the study model because the morphology of the salivary glands in this animal model is relatively similar to that of humans (Wang et al., 1998b).

	MATERIALS & METHODS

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Experimental Animals
Ten inbred, healthy male miniature pigs (6 mos old; weight, 35-50 kg per capita) were obtained from China Agriculture University (Wang et al., 1998b). They were kept under conventional conditions with free access to water and food. The animals were divided randomly into test group (n = 5) and control group (n = 5). The parotid glands of pigs in the test group were ablated bilaterally by retrograde injection of 1% methyl violet (4 mL) to both parotid glands via Stensen’s ducts (Wang et al., 1998a; Li et al., 2001). The nitrate-loading test was performed 3 mos after ablation of the parotid glands. This study was reviewed and approved by the Animal Care and Use Committee of Capital University of Medical Sciences.

Nitrate Background Detection
All animals were fasted for at least 12 hrs before the background levels of nitrate and nitrite were determined. They were given 50 g nitrate-free fodder diluted in 100 mL distilled de-ionized water. General anesthesia (ketamine/xylazine) was administered 30 min later. The mixed saliva was collected for 10 min at 0, 60, 90, and 120 min after 0.5 mg/kg pilocarpine administration (i.m.). The head of the animal was held down, and the drooling saliva was collected into 10-mL vials. A 5-mL quantity of blood was also collected from the precaval vein at 0, 40, 60, 90, and 120 min. The pig was kept in a metabolic cage to facilitate collection of urine samples at 0, 90, 180, and 240 min. All samples were kept at -70°C and analyzed within one month. This experiment was repeated on two separate occasions with an interval of one week.

Nitrate Loading Test
Potassium nitrate (40 mg/kg body weight) was mixed with 50 g nitrate-free fodder diluted in 100 mL distilled de-ionized water, and given to the fasting miniature pigs in both groups. The saliva, serum, and urine samples from these pigs were collected as described above.

Sample Preparation
All samples were collected into sterilized Eppendorf tubes (1 and 10 mL) containing 0.1 and 1.0 mL NaOH (1.0 mol/L) to prevent further reduction of nitrate (Phizackerley and Al-Dabbagh, 1983) and stored at -70°C pending nitrite and nitrate analysis within a month. Before nitrite and nitrate determination, all the samples were de-proteinized by acetone (AR) and filtered through a 0.45-µm filter as described (Chao et al., 1990; Blanco et al., 1995).

Detection of Nitrate and Nitrite
The nitrate and nitrite contents of all samples were determined by high-performance liquid chromatography (HPLC, Model 1050, Hewlett-Packard, Waldbronn, Germany) (Blanco et al., 1995). The injection volume was 6 µL. The column was Hypersil ODS C-18 (250 x 4 mm, 5-µm particle size, Agilent Technologies, Waldbronn, Germany). The HPLC mobile was 1.0 mL/min of KH₂PO₄-H₃PO₄ buffer (0.03 mol/L, pH 3.5). An ultraviolet detector (210 nm) was used to detect nitrate and nitrite at the retention times 2.59 and 3.46 min, respectively. The recovery rate of nitrate in these samples was 95 98%, and of nitrite, 97 100%. The detection limit for nitrate was 0.8 mg/L, and for nitrite, 0.1 mg/L. The first sample of HPLC determination was analyzed in duplicate. If the reproducibility in the duplicated analyses was satisfactory, only one analysis was performed for each of the remaining samples.

Histopathological Examination of Parotid Glands
All animals were killed after the experiments, and their parotid, submandibular, and sublingual glands were examined histopathologically under microscopy.

	RESULTS

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Establishment of Parotid Gland Ablation in a Miniature Pig Model
The morphologically typical serous acini and ductal elements were observed in the parotid glands of the control pigs. The lumina of Stensen’s ducts and ductules were obliterated, acini were replaced by connective tissue, and inflammatory cell infiltrations were present in the parotid glands of the test animals after ablation of the parotid glands. No abnormal findings were seen in the submandibular and sublingual glands of both groups.

To evaluate the effect of the ablation on secretion of saliva, we examined parotid and whole saliva flow rates. Saliva was unobtainable from the parotid glands in the test pigs, even after pilocarpine stimulation. The average flow rate (mL/min) of mixed saliva of the test pigs was reduced to 56.82% of the control group (mean ± SD, 0.75 ± 0.32 vs. 1.32 ± 0.65, P < 0.05, Student’s t test).

Comparison of Nitrate Concentrations [NO₃^–] in Mixed Saliva, Serum, and Urine Samples Before and After KNO₃ Loading
The background [NO₃^–] level in the mixed-saliva samples from the control group was significantly higher than that from the test group (mean ± SD, 14.7 ± 5.2 vs. 6.1 ± 0.9 µg/mL, P < 0.01, by Student’s t test, Fig. 1A). The concentration of nitrate increased to peak values at 60 min after nitrate loading in both groups and then gradually decreased. The salivary [NO₃^–] level in the control group was significantly higher than that in the test group at various time points after nitrate loading (P < 0.05). The recovery speed of salivary [NO₃^–] in the control group seemed to be slower than that in the test group (Fig. 1A).

View larger version (17K): [in this window] [in a new window]   Figure 1. Nitrate concentration (µg/mL) in mixed saliva, serum, and urine of miniature pigs before and after nitrate loading. Data are given as mean ± SD (n = 5). , control group, no KNO3 treatment; •, test group, no KNO3 treatment; , control group, KNO3 treatment; , test group, KNO3 treatment. (A) Mixed saliva samples. Concentration of nitrate in the test group (bilateral ablation of the parotid glands) is significantly lower than that in the control group before and after nitrate loading (p < 0.05). Higher concentration of nitrate was induced by nitrate loading in both groups (p < 0.05). (B) Serum samples and (C) urine samples. Similar concentrations of nitrate were detected in both groups before nitrate loading. Higher concentrations of nitrate were induced by nitrate loading. The concentration of nitrate in the test group was much higher than that in the control group after nitrate loading (P < 0.001).

The background urinary [NO₃^–] level was the same in both groups. The urinary [NO₃^–] level increased to the maximum in both groups at 180 min after nitrate loading. The peak urinary [NO₃^–] level in the test group was much higher than that in the control group (580 ± 69 vs. 260 ± 32 µg/mL, P < 0.001, Fig. 1C).

The Nitrite Concentrations [NO₂^–] in Mixed Saliva, Serum, and Urine Before and After KNO₃ Loading
The background [NO₂^–] level in mixed saliva in the control group was significantly higher than that in the test group (14.5 ± 2.5 vs. 6.5 ± 1.2 µg/mL, P < 0.001). After nitrate loading, the mixed salivary [NO₂^–] level in the control group did not change, while in the test group it increased gradually and reached that of the control group at 120 min after the loading (Fig. 2). Nitrite was undetectable in serum and urine samples from both groups.

View larger version (18K): [in this window] [in a new window]   Figure 2. Nitrite concentration (µg/mL) in mixed saliva of miniature pigs before and after nitrate loading. Data are given as mean ± SD (n = 5). , control group, no KNO3 treatment; •, test group, no KNO3 treatment; , control group, KNO3 treatment; , test group, KNO3 treatment. The background concentration of nitrite in the test group (bilateral ablation of the parotid glands) was significantly lower than that in the control group (P < 0.001). No significant change of nitrite concentration was found in the control group after nitrate loading. Restoration of nitrite concentration to normal level of the control group was found in the test group after nitrate loading.

	DISCUSSION

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Although it has been known for decades that salivary glands secrete nitrate positively, the specific mechanisms involved are not clear, nor are the functions of nitrate and nitrite in saliva known. Hence, we studied the role of the gland in the metabolism of the salivary and systemic nitrate and nitrite using a previously established animal model of parotid gland atrophy in miniature pigs (Wang et al., 1998b; Li et al., 2001).

The present study showed that the average background [NO₃^–] level in mixed saliva in the test animals was much lower than that in the control group. Moreover, this difference should be further amplified, since the average flow rate of mixed saliva in the test group was significantly lower than that in the control one. These results are consistent with findings that the nitrate in mixed saliva is secreted mainly from the parotid glands (Wagner et al., 1983; Cortas and Wakid, 1991), and that retrograde injection of 1% methyl violet ablated parotid glands greatly (Wang et al., 1998b; Li et al., 2001). Ablation of the parotid glands leads to loss of active secretion of nitrate from blood into saliva.

Not surprisingly, after nitrate loading, the [NO₃^–] levels in mixed saliva from both the control and test groups were higher than the background [NO₃^–] level. Moreover, the mixed salivary [NO₃^–] level in the test group was much lower than that in the control group. The [NO₃^–] level in mixed saliva was much higher than the serum [NO₃^–] level in the control group after nitrate loading. Unlike kidneys, which positively absorb water from pro-urine to blood, the parotid glands actively secrete water into saliva (Edgar and O’Mullane, 1996). Thus, higher [NO₃^–] levels in mixed saliva should also result from active secretion of nitrate after nitrate loading. However, the nitrate-loading-induced salivary [NO₃^–] increase in the test group might result from the passive penetration of nitrate from other salivary glands, because the mixed salivary [NO₃^–] level is lower than the serum [NO₃^–] level (Figs. 1A, 1B). We conclude that ablation of the parotid glands destroys the function of active secretion of nitrate from blood into saliva. The consequences of loss of the active secretion of nitrate are unknown.

Ablation of the parotid glands could be observed among the patients with oral or head and neck cancers treated with radiotherapy (Mossman et al., 1982). Hypofunction of the parotid glands can be seen in patients with Sjögren’s syndrome (SS), which is an autoimmune disease that mainly involves salivary and lachrymal glands (Wang et al., 1993). Our clinical study showed that the highest concentration of nitrate was found in parotid saliva, followed by urine, mixed saliva, and serum in healthy subjects (Xia et al., 2003). In patients with SS, the nitrate level in parotid and mixed saliva was markedly decreased but was increased significantly in urine; the concentration of nitrite in mixed saliva was significantly decreased in patients with SS, and increased in patients with sialosis (Xia et al., 2003). In the present study, we observed that the background serum [NO₃^–] levels in both the control and test groups were the same. However, higher serum [NO₃^–] levels were detected in the test group than in the control group after nitrate treatment. These results suggest that loss of the active secretion of nitrate does not interrupt the function of the body to balance the background nitrate, but it does affect the ability of the body to balance blood nitrate after nitrate loading. Nitrate commonly presents in foods, such as vegetables and fruits, and therefore, high blood [NO₃^–] levels could be observed after a meal. It is unknown whether hypofunction of the parotid glands in patients can lead to abnormal levels of blood nitrate. Ablation of the parotid glands might affect the body’s ability to maintain a stable blood nitrate balance.

The urinary background [NO₃^–] level was the same in both groups. Although the urinary [NO₃^–] level was much higher than the serum [NO₃^–] level, it is unlikely that the kidneys secrete nitrate positively. The condensing process of pro-urine in the kidneys might account for the higher urinary [NO₃^–] level. After nitrate loading, the urinary [NO₃^–] level was increased significantly, and a higher [NO₃^–] level was found in the test group than in the control group (Fig. 1C). The higher serum [NO₃^–] level might lead to the higher urinary [NO₃^–] level in the test pigs. These results suggest that restoration of a physiological systemic nitrate level was delayed by parotid ablation and could finally be achieved by increased excretion via urine.

Nitrate can be converted to nitrite by the oral flora (Tannenbaum et al., 1974; McKnight et al., 1999), and nitrite may deter the growth of certain species of bacteria, such as Clostridia (Wolff and Wasserman, 1972). In the present study, the mixed salivary [NO₂^–] level was much higher in the control group than in the test group before nitrate loading. This suggests that salivary [NO₂^–] levels might be [NO₃^–]-dependent. However, salivary [NO₂^–] levels did not change in the control group after nitrate loading. This indicates that the ability of oral flora to convert nitrate to nitrite in oral cavity is limited and easily saturated by exposure to high levels of nitrate. After nitrate loading, the mixed saliva [NO₂^–] returned to the normal level (Fig. 2). This result suggests again that saliva [NO₂^–] is [NO₃^–]-dependent at normal exposure levels of nitrate.

Saliva [NO₂^–] are increased following incubation of mixed saliva at 37°C, but not increased when the saliva was first sterilized by filtration (Ishiwata et al., 1975b). This indicates that only microorganisms presenting in mixed saliva might contribute to the conversion of nitrate to nitrite. Nitrate reductase of those microorganisms is the known enzyme responsible for converting nitrate to nitrite in saliva. It will be useful to compare activity of nitrate reductase in mixed saliva collected from both groups.

	ACKNOWLEDGMENTS

 
This study was supported by the National Natural Science Foundation of China (Grants 30125042, 30271400) and by Beijing Grant 7002020. The authors express sincere gratitude to Dr. Bruce Baum, NIDCR, NIH, USA, and Prof. L.P. Samaranayake, Hong Kong University, for critically reading the manuscript. The authors also express thanks to Ms. Fengying Dong, Experimental Animal Units, Faculty of Stomatology, and Ms. Xiuyu Cui, Lin Li, Institute of Nerve Science, Capital University of Medical Sciences, for their technical assistance. This work is based on a thesis submitted to the Faculty of Stomatology, Capital University of Medical Sciences, in partial fulfillment of MS degree requirements.

Received for publication November 5, 2001. Revision received October 24, 2002. Accepted for publication November 6, 2002.

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Journal of Dental Research, Vol. 82, No. 2, 101-105 (2003)
DOI: 10.1177/154405910308200205


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