Plasma Asymmetrıc Dımethylargınıne (ADMA) and Nıtrıc Oxıde (NO) Levels ın Patıents wıth Chronıc Pansınusıtıs

Abstract

Chronic pansinusitis is a mucosal inflammation of the nose and all paranasal sinuses with severe inflammation of the upper airways. Asymmetric dimethylarginine (ADMA) is associated with oxidative stress. In this study, we aimed to examine the plasma levels and importance of ADMA and nitric oxide (NO) in patients with chronic pansinusitis. The study was conducted with a total of 64 patients. The study group included a total of 40 patients with chronic pansinusitis. (18 females, 22 males) (mean age 32.27 ± 10.02). The control group consisted of 24 patients (11 females and 13 males). The mean age of the patients in the control group was 31.35 ± 6.05 years. Nasal endoscopic examinations were performed in patients with a history of chronic pansinusitis and symptoms of chronic pansinusitis. Later, the diagnosis of chronic pansinusitis was confirmed with coronal paranasal sinus Computed tomography scans. Plasma ADMA levels were measured by ELISA method and NO levels were measured by Griess method. Plasma ADMA and NO levels of the patients and healthy volunteers were measured and the mean plasma levels of the two groups were compared. ADMA levels were significantly higher in the group with chronic pansinusitis compared to the control group (1.23 ± 0.41 μM and 0.28 ± 0.06 μM, respectively) (p < 0.001), while NO levels were significantly lower in the patient group compared to the control group (7.06 ± 1.07 μM and 12.25 ± 0.95, μM, respectively) (p < 0.001). Our results show that the increase in ADMA levels and the decrease in NO levels are associated with chronic pansinusitis. According to these results, increased plasma levels of ADMA in chronic pansinusitis may be useful in clinical use as a sign of increased oxidative stress.

Introduction

Chronic pansinusitis is a persistent mucosal inflammation of the nose and all paranasal sinuses with severe inflammation of the upper airways and is a clinical picture that lasts more than 12 weeks with or without nasal polyp. Chronic rhinosinusitis is also economic burden on patients due to its high prevalence as well as its negative effects on health and quality of life [1]. The pathogenesis of chronic sinusitis is multifactorial. Inhaled allergens, bacterial or viral infections and hypoxia play a role in the formation of chronic sinusitis by causing impairment in the mucosal epithelial defense of the nose and sinuses. The pathology of mucosal epithelial defense in chronic sinusitis is characterized by a wide range of immunological mechanisms such as the production of eicosanoids and IgE, (TGF-β) and an increase in T cell signaling, eosinophilic response to neutrophilic inflammation [2]. Disruption of the delicate balance between the normal production of reactive oxygen species such as superoxide anion radical and hydroxyradical and protective antioxidants also causes chronic sinusitis [3].

NO is metabolized from L-arginine in the presence of nitric oxide synthase (NOS). Presence of NO in the nasal airway was first identified by Alving et al. [4]. NO is found in very high concentrations in the paranasal sinus epithelium and contributes to the formation of a sterile sinus environment [5]. NOS is also abundant in the parasympathetic neural tissues in the pterygopalatine ganglion and in the nasal mucosa. [6] It is well known that there are three different isoforms of NOS; neuronal NOS-1 (nNOS), inducible NOS-2 (iNOS) and endothelial NOS-3 (eNOS). iNOS is released by mast cells during inflammation and its activity greatly increases [7]. The excess NO combines with superoxide radicals and turns into peroxynitrite. During hypoxia, NO has an important role in tissue homeostasis. NO has vasodilator, bacteriostatic and antiviral properties in the upper respiratory tract also it is a potent stimulator for ciliary activity [8–10] Serum ADMA is an endogenous NOS inhibitor that competes with arginine and it affects the bioavailability of NO. It leads to a decrease in NO levels and it has a negative impact on immune function [11]. High ADMA concentrations can directly induce oxidative stress and cell apoptosis and participate in inflammation reactions [12]. Dimethylaminohydrolase (DDAH) enzyme metabolizes most of ADMA. DDAH enzyme activity is extremely sensitive to oxidative stress. DDAH activity decreases as a result of inflammation and oxidative stress. With the inactivation of the DDAH enzyme, ADMA elimination is impaired and ADMA levels increase while NO production decreases. DDAH activity also decreases with oxidative stress induced by TNF-α [13]. Normal serum ADMA levels are in the range of 0.25–0.92 μM [14].

Medical conditions with high plasma ADMA levels: hypercholesterolemia, hypertriglyceridemia, hyperhomocysteinemia, atherosclerosis, insulin resistance, Type 2 diabetes mellitus, hypertension, preeclampsia, pulmonary hypertension, coronary artery disease, congestive heart failure, peripheral heart failure, erectile dysfunction, schizophrenia, stroke, aging and Alzheimer's disease [15–17]

ADMA is a useful marker and tool to detect oxidative stress. ADMA blood level increases during oxidative stress [18]. Proteolysis activiy is enhanced in case of severe inflammation. Increased proteolysis, decreased elimination leads to ADMA accumulation [19].

We examined the relationship between ADMA and NO plasma levels and chronic pansinusitis with a clinical picture indicating severe inflammation. According to our literature research, this study is the first study investigating serum ADMA and NO levels in chronic pansinusitis.

Material and Methods

The protocol of the study was approved by Tekirdağ Namık Kemal University Faculty of Medicine Ethics Committee (protocol number 2019.149.09.09). All participants provided informed consent. This study was conducted between September 2019 and July 2020. Clinical samples were obtained from Private Çorlu Reyap Hospital Otorhinolaryngology Head and Neck Surgery Department. Laboratory studies were carried out in Tekirdağ Namık Kemal University Faculty of Medicine Biochemistry Laboratory.

A total of 64 patients were included in this study 40 patients (18 females, 22 males) had a history of chronic pansinusitis and 24 (11 females and 13 males) were volunteers in our outpatient clinic at the Çorlu Private Reyap Hospital. Chronic pansinusitis patients between the ages of 22–44 who were referred to the ENT outpatient clinic were consecutively included in this study. In our study, Body mass index (BMI) was 21.3 in the patient group and 22.5 in the control group. Patients presented with chronic sinusitis symptoms (runny nose, headache) for at least 3 months and had not used antibiotics in the last 3 months. Chronic pansinusitis was diagnosed and confirmed by CT scans of paranasal sinuses. Blood serum levels of ADMA and NO were compared.

Inclusion criteria: Diagnosis and treatment criteria were taken as inclusion criteria. Patients with chronic pansinusitis symptoms were diagnosed with coronal paranasal sinus CT. The control group consisted of patients with coronal paranasal sinus CT who had no inflammation in the sinuses and underwent septoplasty.

Exclusion criteria: Being < 18 years of age and > 45 years of age, pregnant, smoking, alcohol consumers. Having chronic hematologic, renal, hepatic, endocrinal, neoplastic, inflammatory or vascular diseases (cardiac, cerebral and/or peripheral). Patients using (antibiotics, antiepileptic, antihypertensive, antidiabetic, anticoagulant, lipid lowering drugs, vitamin supplements, oral contraceptives, antioxidants) in last 3 months.

Collection of Blood Serum Samples

On the first day of admission to the otorhinolaryngology outpatient clinic, blood samples were taken from the patients in 10 mm tubes with a red cap which do not consist gel. Samples were centrifuged at 1,500 xg for 10 min. Serum samples used in determination of ADMA and NO levels were stored at − 80 °C until analysis.

Measurement Method of ADMA Levels

ADMA plasma concentrations were tested using the human ADMA ELISA kit (Cat no. E1887Hu, Bioassay Technology Laboratory, Shanghai, China). ADMA level was expressed in μM. The plate is coated with human ADMA antibody. ADMA in added sample binds to ADMA antibodies. The biotinylated ADMA antibody is then added and binds to the ADMA in the sample. When Streptavidin-HRP is added it binds to the biotinylated ADMA antibody. Unbound streptavidin-HRP is removed by the washing step after incubation. When the substrate solution is added, color is formed in proportion to the ADMA concentration. This reaction is halted with acidic stop solution. Absorbance is measured at 450 nm.

Measurement Method of NO Levels

Plasma NO concentration was measured using Cayman Chemical (Cayman Chemical Company, Ann Arbor, MI. USA) commercial ELISA kit. NO levels were expressed in μM. There are two steps in measuring NO concentration. In the first step, nitrate is converted to nitrite by using nitrate reductase enzyme. In the second step, the purple colored azo compound is formed by adding Griess reagent. The nitrite concentration is determined by measuring this compound at 540 nm [20].

Statistical Methodology

PASW Statistics 18 of the Windows program was used for data entry and statistical analysis. The mean value and standard deviation (or median, minimum–maximum value) were used to express the results. Normality has been assessed. Student’s t-test (or Mann Whitney U test) was used to compare the two groups. Statistical significance was accepted as p < 0.001.

Results

40 patients diagnosed with chronic pansinusitis were included in this study; 22 men and 18 women. The mean age of the patients was 32.27 ± 10.02 years. 24 patients were included in the control group. The mean age of the control group was 31.35 ± 6.05 years. We found that blood serum ADMA results were significantly increased in the patient group. (1.23 ± 0.41 μM and 0.28 ± 0.06 μM, respectively) (p < 0.001) (Fig. 1). And NO levels were significantly lower in the patient group than controls (7.06 ± 1.07 μM and 12.25 ± 0.95 μM, respectively) (p< 0.001) (Fig. 2) (Table 1).

Fig. 1.

ADMA measurements in control and chronic pansinusitis patients

Fig. 2.

NO measurements in patients with control and chronic pansinusitis

Table 1.

Biochemistry parameters of patients and controls

	Group	n	Mean ± SD	Median (min–max)	p value
ADMA (µM)	Control	24	0,28 ± 0,06	0,29 (0,13–0,15)	p < 0,001
	Study	40	1,23 ± 0,41	1,37 (0,39–1,67)
NO (µM)	Control	24	12,25 ± 0,95	12,30 (10,43–13,89)	p < 0,001
	Study	40	7,06 ± 1,07	7,21 (4,22–8,98)

Discussion

In our study, we detected that plasma ADMA levels are higher and NO levels are lower in chronic pansinusitis patients. ADMA levels were significantly higher in the group with chronic pansinusitis compared to the control group (1.23 ± 0.41 μM and 0.28 ± 0.06 μM, respectively) ( p< 0.001) (Fig. 1). This may explain the increased oxidative stress in chronic pansinusitis. According to literature research we have done so far, our study is the first to reveal an increase in plasma ADMA levels associated with a decrease in NO production in patients with chronic pansinusitis.

In a previous 24-year follow-up study, it was reported that increased ADMA levels were associated with the incidence of fatal and nonfatal myocardial infarction and stroke [21].

However, it has been reported in previous studies that chronic rhinosinusitis may lead to an increase in cardiovascular diseases. It has been reported that patients with chronic rhinosinusitis may have a higher risk of myocardial infarction [22]. Increased ADMA levels have been associated with proinflammatory conditions in previous studies, and an increased risk of mortality has been reported in septic patients [23].

In our study, we found that plasma NO levels in patients with chronic pansinusitis were significantly lower than in the control group (7.06 ± 1.07 μM and 12.25 ± 0.95 μM, respectively) (p < 0.001) (Fig. 2).

In a previous study, it has been reported that enlargement of the ostiums in sinus surgery reduces the NO concentration in the sinus and this may lead to a decrease in protection against infections [24]. This may be an explanatory factor for some unsuccessful results in sinus surgery. It has been reported that increased ADMA concentrations can be considered as a marker for predicting cardiovascular mortality and morbidity [25]. Studies have suggested that ADMA is not only a disease outcome or prediction tool, but also a new potential therapeutic target. Although therapeutic tools targeting ADMA have been found in cardiovascular diseases, its effects in chronic pansinusitis are unknown [26].

Our results show the importance of increased plasma ADMA levels in chronic pansinusitis. Increased ADMA levels may be a new clinical and experimental risk marker for chronic pansinusitis. In our study, we found significantly higher plasma ADMA levels in patients with chronic pansinusitis compared to the control group, and significantly lower NO levels. (p < 0.001).

It has been reported that the increase in plasma ADMA levels can be accepted as a risk factor for coronary artery disease along with other risk factors such as hypertension, hypercholesterolemia, diabetes mellitus and smoking [27].

In a previous study on ADMA levels, a positive correlation between ADMA and BMI was found [28]. However another study suggested that, decrease in ADMA levels was associated with increase in BMI [14]. In our study, the BMI was 21.3 in the patient group and 22.5 in the control group. We did not find any correlation between BMI and ADMA. ADMA can also be detected in urine [29]. In our study, we determined the blood plasma ADMA levels.

To date, no reference values for ADMA have been established for patients under 18 years of age. Therefore, we did not include patients younger than 18 years of age in our study. Previous studies have reported an increase in ADMA blood levels in elderly patients [30]. Elderly patients were not included in our study. In our study, the female-male ratio 18 female, 22 male and, the mean age was 32.27 ± 10.02 in the patient group. In the control group, on the other hand, female-male ratios 11 females 1 male and, the mean age was 31.35 ± 6.05 years. There was no statistically significant difference between the two groups in terms of ADMA between gender. (p > 0.05)

In a previous study conducted with healthy people, ADMA levels showed no statistically significant difference between men and women [31]. In our study, we found that plasma NO levels were significantly lower in the chronic pansinusitis group compared to the controls (7.06 ± 1.07 μM and 12.25 ± 0.95 μM, respectively) (p < 0.001). However, previous studies have suggested that NO levels are reduced in the breathing air of chronic rhinosinusitis patients compared to those with healthy sinuses [32]. Nasal NO can play a role as an inflammatory marker in the upper airway [33].

In addition, ADMA inhibits all three forms of the NOS enzyme competitively, also reversing this inhibition with high concentrations of L-Arginine [34]. Antioxidants, folic acid, vitamin B6 and B12 have been shown to reduce ADMA levels in previous studies [35, 36] It was reported that exercise reduced ADMA by reducing oxidative stress [37]. L-citrulline supplementation is well tolerated and can be studied in high-risk patients with elevated ADMA [38]. However, no specific ADMA lowering agent is yet available.

In our study, we observed a significant increase in plasma ADMA concentration in chronic pansinusitis patients compared to healthy controls. Our results have demonstrated a possible role of NO-associated ADMA levels in the pathogenesis of chronic pansinusitis. Measuring ADMA levels will have important contributions to early diagnosis of chronic pansinusitis. New treatment approaches that will reduce the level of ADMA and the use of ADMA as a marker in diagnosis and follow-up care will be subject to future studies. There are some factors that limit our study. First of all, our results are observational and should not be interpreted in terms of causality. Because cross-sectional studies do not allow causal relationships to be defined.

Conclusion

These data revealed increased plasma ADMA levels in patients with chronic pansinusitis associated with a decrease in NO production. Increased oxidative stress in chronic pansinusitis plays an important role in sinus function and changes in the sinus mucosa after obstruction of sinus ostium. We think that chronic pansinusitis with severe inflammation increases the plasma ADMA level by increasing oxidative stress. ADMA plasma levels may be a new clinical and experimental biomarker related to chronic pansinusitis. New treatment approaches that will reduce the level of ADMA. The use of ADMA as a laboratory test and a marker in the follow-up of the treatment of this disease will also be a subject for future studies. We propose further studies on the effect of the oxidative stress marker ADMA elevation on the outcome of the antioxidant administration as an adjuvant therapy for chronic pansinusitis. More large-scale clinical studies are needed in the future.

Funding

The present study has been supported by Istanbul Rumeli University, through the Scientific Research Project no: 2019010.

Declaration

Conflict of ınterest

The authors declare that they have no conflict of interest.