Evaluation of Salivary Alkaline Phosphatase and Glutathione Peroxidase Levels in Diabetic and Nondiabetic Participants With and Without Smoking Habits: A Case-Control Study.

Background: The majority of the current evidence suggests that tobacco smoking increases the risk of diabetes. Salivary alkaline phosphatase (ALP) and glutathione peroxidase (GSHPx) considered a biomarker to detect various oral diseases. Several studies suggest that smoking habits tend to alter ALP and GSHPx levels. However, at present, there is no much information about these enzymes in smokers with diabetes. Hence, the study aimed to evaluate the status of salivary ALP and GSHPx levels in diabetic and nondiabetic participants with and without smoking habits. Materials and
Methods: This case-control study was approved by the Institutional Ethical Committee. A total of 60 male participants between the age group 35-50 years were recruited. Informed consent was obtained from participants. Participants were categorized into four groups: Group I - Smokers with diabetes (n = 15), Group II - Smokers without diabetes (n = 15), Group III - Nonsmoker with diabetes (n = 15), and Group IV - Nonsmoker without diabetes (n = 15). Salivary ALP levels and GSHPx activity were measured by colorimetric assay. Data were compared between groups using the one-way analysis of variance, followed by a Bonferroni post-hoc test.
Results: Nonsmoker diabetic participants demonstrated significantly higher ALP levels as compared to other groups (P < 0.05). We observed significantly lower levels of ALP in smokers with diabetes (P < 0.05). We observed a significant decrease in GSHPx activity in smokers with diabetes compared to all other groups (P < 0.05). Conclusions: Salivary ALP can be used as a clinical biomarker to be correlated for evaluating diabetes. GSHPx activity can be used to understand the response of supplementation therapy in smokers with diabetes. Copyright:

INTRODUCTION

It is well known that tobacco smoking increases the risk of oral diseases and metabolic disorders such as diabetes mellitus (DM). There is convincing evidence that smokers are 30%–40% likely to develop diabetes than nonsmokers.[123] Smoking influences oral health and its negative effect can be assessed in the saliva, considered an excellent screening and diagnostic tool in dentistry.[4] Saliva acts as an indicator and helps to identify immunological, inflammatory, endocrine, and metabolic biomarkers.[56] Several enzymes and antioxidants in saliva are known to offer protection against oxidative stress and cellular damage.[7] Alkaline phosphatase (ALP) is a hydrolase intracellular enzyme that plays a key role in cellular metabolism. ALP is a glycoprotein produced within the periodontium and gingival crevice.[468] ALP is secreted by polymorphonuclear neutrophils, osteoblast, and periodontal ligament fibroblast at the time of inflammation, bone formation, and periodontal regeneration, respectively.[9] Owing to diabetes, damaged cells of periodontal tissues consistently release ALP into the gingival crevicular fluid (GCF) and saliva.[46] Previous studies have shown significantly increased levels of GCF, serum, and salivary ALP in chronic periodontitis,[6891011] smokers and tobacco chewers with diabetes,[4911121314] and oral malignant diseases.[41113] Among these, few studies have been conducted, mostly in smokers with diabetes and periodontitis.[41314] Hence, the status of ALP in smokers with diabetes alone is relatively sparse. In DM, an increase in the free radicals production or decrease in the antioxidant activity leads to oxidative stress.[15] Glutathione peroxidase (GSHPx) is a cytosolic antioxidant enzyme that prevents reactive oxygen species and oxidative damage in cells.[71617] Several reports show inconsistency in the GSHPx levels in diabetes.[1819202122] Therefore, the purpose of this study was to evaluate the status of salivary ALP and GSHPx levels in diabetic and nondiabetic participants with and without smoking habits.

MATERIALS AND METHODS

This case–control study was conducted at Central Research Laboratory, Rajas Dental College and Hospital. The study was conducted in accordance with the ethical norms of the institutional ethical committee. Participants with other systemic and oral diseases, tobacco chewers, alcoholics, and premalignant lesions were excluded from the study. A total of 60 male participants between the age group 35–50 years were recruited. The study was verbally explained and informed consent was obtained from all participants. Participants were categorized into four groups: Group I – Smokers with diabetes (n = 15), Group II – Smokers without diabetes (n = 15), Group III – Nonsmoker with diabetes (n = 15), and Group IV – Nonsmoker without diabetes (n = 15). 5 mL of unstimulated saliva sample was collected from all participants. The study participants were instructed not to consume food for 2 h before saliva collection to avoid diurnal variation. Participants were instructed to collect saliva on the floor of the mouth and then spit it into a sterile sample collection container.

Measurement of alkaline phosphatase in saliva

Salivary ALP was measured using AUTOSPAN® kit as per the manufacturer's protocol (Arkray Healthcare Pvt. Ltd., India). At pH 10.3, ALP catalyzes the hydrolysis of colorless p-nitrophenyl phosphate to yellow-colored p-nitrophenol and phosphate. Briefly, 20 μL of saliva was mixed with the working reagent. The absorbance was read after 30 s for regular intervals at 405 nm in the colorimeter. The mean absorbance change per minute was recorded.

Determination of glutathione peroxidase levels in saliva

GSHPx was measured as described previously by Rotruck et al. (1973).[23] Briefly, 200 μL of serum mixed with an equal amount of 0.8 mM EDTA, 10 mM sodium azide, 1 mM reduced glutathione (GSH), 2.5 mM H2O2, and 0.32M phosphate buffer (pH 7.0) and incubated at 37°C for 10 min. The reaction was arrested by the addition of 500 μL of 10% (w/v) trichloroacetic acid and centrifuged at 3000 rpm. Then, 500 μL of the supernatant was added with 3.0 mL of 0.33 mM phosphate solution and 1.0 mL of 0.6 mM 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB). Immediately, the formation of yellow color was read at 420 nm using a colorimeter. Phosphate solution and DTNB reagent alone served as blank. A standard is used to compare test values. The activity of GSHPx is expressed as μg of GSH utilized/min/mg protein. GSHPx levels in plasma and saliva samples were also measured using the same procedure.

Statistical analysis

Statistical analysis was performed using SPSS software (v25, Inc., Chicago, IL, USA). The mean values of enzyme levels were compared between groups using the one-way analysis of variance (ANOVA), followed by a Bonferroni post-hoc test. P < 0.05 was considered statistically significant.

RESULTS

Overall, 60 male patients were studied. The mean age of the participants among study groups is summarized in Table 1.

Table 1

Mean age of study participants

Group	n	Minimum	Maximum	Mean	SD
Smokers with diabetes	15	33.00	50.00	43.13	6.34
Smokers without diabetes	15	33.00	50.00	42.06	5.33
Nonsmokers with diabetes	15	35.00	50.00	42.40	4.57
Nonsmokers without diabetes	15	35.00	49.00	41.46	4.58

SD: Standard deviation

Table 2 depicts a statistically significant difference in ALP levels between groups (P < 0.001). We observed a significant increase in ALP levels in nonsmokers with diabetes as compared to all other groups. We observed a decrease in ALP levels in smokers with diabetes. The mean of the ALP levels of nonsmokers without diabetes was found as close to the nonsmokers without diabetes.

Table 2

Comparison of alkaline phosphatase levels between groups (n=60)

ALP levels	n	Mean	SD	P
Smokers with diabetes	15	25.91	8.95	0.001*
Smokers without diabetes	15	39.02	13.65
Nonsmokers with diabetes	15	41.10	14.06
Nonsmokers without diabetes	15	41.91	6.27

*Significant P<0.05 (calculated using one-way ANOVA). SD: Standard deviation, ALP: Alkaline phosphatase, ANOVA: Analysis of variance

As shown in Table 3, the mean difference of ALP levels was statistically significant in smokers with diabetes when compared to other groups. Surprisingly, the mean difference was higher (P = 0.002) in nonsmokers without diabetes. Subsequently, (P = 0.003) in nonsmokers with diabetes and (P = 0.014) smokers without diabetes.

Table 3

Post hoc analysis on alkaline phosphatase levels

ALP levels	Mean difference	P
Smokers with diabetes
Smokers without diabetes	13.10	0.014*
Nonsmokers with diabetes	15.18	0.003*
Nonsmokers without diabetes	16.00	0.002*
Smokers without diabetes
Nonsmokers with diabetes	2.07	1.000
Nonsmokers without diabetes	2.89	1.000
Nonsmokers with diabetes
Nonsmokers without diabetes	0.81	1.000

*Significant P<0.05 (Post hoc analysis using Bonferroni after adjusting for multiple variables). ALP: Alkaline phosphatase

Table 4 shows a statistically significant difference in GSHPx levels between groups (P < 0.05). We observed a significant decrease in GSHPx levels in smokers with diabetes as compared to all other groups. We observed an increase in GSHPx levels in nonsmokers without diabetes.

Table 4

Comparison of glutathione peroxidase levels between groups (n=60)

GSHPx levels	n	Mean	SD	P
Smokers with diabetes	15	0.42	0.49	0.052*
Smokers without diabetes	15	0.55	0.27
Nonsmokers with diabetes	15	0.92	0.72
Nonsmokers without diabetes	15	1.02	0.98

*Significant P<0.05 (calculated using one-way ANOVA). GSHPx: Glutathione peroxidase, ANOVA: Analysis of variance, SD: Standard deviation

As summarized in Table 5, the mean difference of GSHPx levels was statistically insignificant in smokers with diabetes compared to other groups.

Table 5

Post hoc analysis on glutathione peroxidase levels

GSHPx levels	Mean difference	P
Smokers with diabetes
Smokers without diabetes	0.13	1.000
Nonsmokers with diabetes	0.49	0.289
Nonsmokers without diabetes	0.60	0.106
Smokers without diabetes
Nonsmokers with diabetes	0.36	0.856
Nonsmokers without diabetes	0.46	0.367
Nonsmokers with diabetes
Nonsmokers without diabetes	0.10	1.000

*Significant P<0.05 (Post hoc analysis using Bonferroni after adjusting for multiple variables). GSHPx: Glutathione peroxidase

DISCUSSION

In this study, we have investigated the salivary ALP and GSHPx activity in diabetic and nondiabetic with and without smoking habits. It has been shown previously that tobacco smoking contributes to the development of diabetes, a metabolic disorder that causes increased blood sugar levels which leads to severe health complications in the body. Exposure to elevated levels of nicotine in tobacco tends to lower insulin levels.[24] Almost all of the previous studies have shown increased ALP levels due to inflammation in smokers with diabetes.[41314] However, the trend is contradictory to the findings of our study revealed lower ALP levels in smokers with diabetes. This may be due to the combined effect of the smoking habit in diabetic conditions have suppressed the immune system that reduced the production of ALP in smokers with diabetes. On the other hand, we observed higher levels of ALP in nonsmokers with diabetes. This observation is in agreement with the findings of Sridharan et al.,[11] found a significant correlation of ALP in diabetic participants. These observations suggest that inflammation and chronic disease conditions led to the higher accumulation of ALP in diabetics. Furthermore, alteration in the ALP is owing to the lack of adequate insulin synthesis in diabetics.[14] Antioxidant enzymes play a crucial role in the host defense system.[7] The salivary antioxidant enzyme GSHPx offers protection against oxidative stress induced by toxic chemicals in smoking.[25] Most importantly, increased oxidative stress impairs the host antioxidant defense system.[22] We observed a significant decrease in GSHPx activity in smokers with diabetics as compared to other groups. This observation is in agreement with the earlier studies that also found a notable alteration in GSHPx activity in diabetics[26] and smokers.[2728] Taken together, alterations in salivary ALP levels and reduction in GSHPx activity cause inflammation and destruction in healthy tissue. However, there are few limitations to our study. We have included a small sample size. Further investigation in a larger cohort is necessary to validate our findings.

CONCLUSIONS

In summary, these observations led us to conclude that salivary ALP can be used as a clinical biomarker that needs to be correlated in the evaluation of diabetic status. Antioxidant enzyme GSHPx offers cellular and tissue protection from inflammation and oxidative damage. Hence, GSHPx activity can be used to understand the response of supplementation therapy in smokers with diabetes.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.