| Abstract|| |
Background: Oral cytology studies have claimed that cytoplasmic Periodic Acid Schiff (PAS) positivity in type-2 diabetics is due to glycogen content. But, it can also be due to mucin and glycoconjugates. Aim: 1. To confirm that cytoplasmic PAS positivity in type-2 diabetics is due to glycogen using diastase. 2. To know the effect of diabetes by determining the number of glycogen-containing cells in the smear. 3. To assess the impact of duration of diabetes based on PAS staining of cells. 4. To correlate between random blood glucose level and the number of PAS-positive cells. Materials and Methods: Study population comprised 45 individuals with 30 type-2 diabetics as case group (Group I < 5 years duration; Group II > 5 years duration) and 15 healthy volunteers (age and gender-matched) as control. For all subjects, random blood glucose was estimated and two cytosmears were obtained. The smears were stained with PAS and PAS-diastase stains (PAS-D). Staining intensity was documented as score 1 (mild-to-moderate) and score 2 (moderate-to-intense) and data obtained were statistically analyzed in SPSS version 16.0. Results: Mann-Whitney U test revealed that in diabetics cytoplasmic PAS positivity is because of glycogen (P < 0.05). There is an increase in the number of glycogen-containing cells (P < 0.05) in diabetics. The duration of diabetes had less impact on intracellular glycogen accumulation (P > 0.05). Spearman's correlation test revealed no significant correlation (P > 0.05) between random blood glucose and a number of PAS-positive cells. Conclusion: PAS positivity is because of intracellular glycogen accumulation in type-2 diabetics. It can convey the glycaemic status of an individual in the recent past, thus a beneficial role in screening and therapeutic monitoring.
Keywords: Cytology, diabetes, diastase, glucose levels, glycogen, periodic acid Schiff
|How to cite this article:|
Ravikumar S S, Menaka T R, Vasupradha G, Dhivya K, Dinakaran J, Saranya V. Cytological intracellular glycogen evaluation using PAS and PAS-D stains to correlate plasma glucose in diabetics. Indian J Dent Res 2019;30:703-7
|How to cite this URL:|
Ravikumar S S, Menaka T R, Vasupradha G, Dhivya K, Dinakaran J, Saranya V. Cytological intracellular glycogen evaluation using PAS and PAS-D stains to correlate plasma glucose in diabetics. Indian J Dent Res [serial online] 2019 [cited 2020 Apr 2];30:703-7. Available from: http://www.ijdr.in/text.asp?2019/30/5/703/273450
| Introduction|| |
Diabetes mellitus (DM), an endocrine disorder is an evergrowing burden worldwide. Type-2 diabetes (noninsulin-dependent) occurring either because of resistance to the action of insulin on cells or a relative deficiency of insulin make-up about 90% of cases of diabetes in India. Glycogen is the primary storage form of glucose and is known to increase intracellularly in diabetics.
Many oral exfoliative cytology studies in type-2 diabetics have shown increased intracellular accumulation of glycogen through PAS staining as glycogen is PAS-positive. Since PAS positivity could also be due to mucin and glycoconjugates, we decided to perform a pilot study to confirm the presence of glycogen through diastase digestion.
| Aims and Objectives|| |
The aims and objectives of the present study are:
- To confirm that the PAS staining in exfoliated buccal cells of type-2 diabetics is due to glycogen using diastase digestion.
- To ascertain the impact of type-2 diabetes on a number of glycogen-containing cells by comparing the PAS staining between the case group and control group.
- To assess the impact of duration of diabetes on intracellular glycogen accumulation by comparing the intensity of PAS staining among type-2 diabetics.
- To find the correlation between random blood-glucose level and the number of PAS-positive cells in the study population.
| Materials and Methods|| |
Institutional ethical clearance was obtained to perform this study. The present case-control study comprised of 45 individuals with 30 type-2 diabetic patients as case group and 15 healthy volunteers (age and gender-matched) as a control group. Based on the duration of diabetes, cases with less than 5 years of diabetes were considered as group I and more than 5 years of diabetes as group II. Most of the diabetics were on oral hypoglycaemics with two of them taking insulin. Patients with frank oral lesions and other systemic disorders were excluded from the study.
After obtaining the informed consent, capillary blood glucose was measured by the finger-prick method using the glucometer (One Touch Ultra-Lifescan Inc., USA) based on the principle of glucose oxidase-peroxidase method.
A thorough examination of the hard and soft tissues of the oral cavity was done and the subjects were asked to rinse their mouth with water. After this, a disposable wooden spatula was used with a gentle scrap motion to obtain cell scrapings from the buccal mucosa. Two cytological smears were prepared for each individual, fixed immediately in 95% ethanol and subjected to PAS (Nice's chemicals (P) Limited) and PAS-diastase (PAS-D) stains, respectively. The freshly prepared solution of diastase using 0.25 g of diastase (Loba Chemie Pvt ltd) in 50 ml distilled water was used. For PAS-D staining, the smears were first incubated in diastase solution for 1 hour at room temperature and then stained with PAS stain.
All cytological smears were analyzed and scored. Mild to moderately stained cells were scored as 1 [Figure 1]a and moderate to intensely stained cells were scored as 2 [Figure 1]b. The number of PAS-positive cells (magenta-colored cells) in 100 clearly defined cells were counted by moving the microscope stage from left to right, then down and across to avoid counting the same cell again. Cellular clumps were avoided while counting the PAS-positive cells.
|Figure 1: (a) Mild-to-moderate stained cells (score 1) (PAS, 40×). (b) Moderate-to-intense stained cells (score 2) (PAS, 40×)|
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The data obtained were subjected to statistical analysis (SPSS version 16.0). Descriptive statistics were obtained. Mann-Whitney U test and Spearman's correlation were performed. P value less than 0.05 was taken as significant.
| Results|| |
In this study, the case group included 13 males and 17 females with an age range of 36–73 years. Control group comprised of 6 males and 9 females with an age range of 33–72 years. Random blood glucose (RBG) values in the case group ranged 102–444 mg/dl and in the control group, it was 97 mg/dl–152 mg/dl.
To confirm that the PAS positivity in exfoliated buccal cells of type-2 diabetics is due to the presence of glycogen, we compared the PAS and PAS-D scores in the study population and we found a significant difference (P < 0.05) in the number of PAS-positive cells in diabetics using Mann-Whitney U test [Table 1].
|Table 1: Comparison of PAS-positive cells in PAS and PAS-D stain in case and control group using Mann-Whitney U-test|
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To know the effect of type-2 diabetes on the number of glycogen-containing cells, we compared the PAS staining between the cases and controls. We found a statistically significant increase (P < 0.05) in a number of PAS-positive cells in case group using the Mann-Whitney U test [Table 2].
|Table 2: Comparison of PAS staining between the case group and control group using Mann-Whitney U-test|
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To assess the impact of duration of diabetes on intracellular glycogen accumulation, we compared the number of PAS-positive cells (score 1 and 2) in case groups (Group I and II) but we did not find any significant difference (P > 0.05) using Mann-Whitney U test [Table 3].
|Table 3: To compare the intensity of PAS staining among the cases (group I and II) to assess the impact of duration of diabetes using the Mann-Whitney U-test|
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To know whether we can estimate the blood-glucose level with the number of PAS-positive cells, we did a Spearman's correlation test between random blood-glucose level and the number of PAS-positive cells. However, we did not find any significant correlation in the case group and control group [Table 4].
|Table 4: Correlation between random blood glucose and the total PAS-positive cells in case and control group using Spearman's correlation (r)|
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| Discussion|| |
A fasting plasma-glucose level >126 mg/dl (7.0 mmol/L) meets the threshold for the diagnosis of diabetes. DM is clinically diagnosed when random blood glucose exceeds 140 mg/dl. The normal blood-glucose level ranges between 70 mg/dl to 130 mg/dl. Glycogen, a large macromolecule joined by α(1,4) glycosidic linkage, is a major storage form of glucose and is usually present as granules in the cell cytoplasm [Figure 2]. Good coordination is essential between glycogen synthesis and breakdown to maintain blood glucose levels.
Normally, grains of glycogen is seen in the spinous layer and superficial layers of non-keratinized oral epithelium. Glycogen is not stored in the basal layers of epithelium because of higher mitotic activity and greater consumption of glucose by cells of this layer. The entry of glucose into the cells is facilitated by two specific transport system: insulin-independent and insulin-dependent transport system of glucose. Six glucose transporters (GLUT-1–GLUT-5 and GLUT-7) have been identified but they are tissue-specific. In the cell cytoplasm, glycogenesis occurs with glucose entering the cell through GLUT-1 and in glycogenolysis that involves the breakdown of glycogen to monomeric glucose, glucose exits the cell through the GLUT-2 transporter.
PAS technique detects a varied number of mucosubstances such as glycogen, mucins, and glycoproteins and is based upon the reactivity of free aldehyde groups within carbohydrates with the Schiff reagent to form magenta-colored end product [Figure 3]a. Diastase is α – amylase which catalyzes the hydrolysis of the glycosidic bonds of glycogen and the breakdown of the large glycogen molecules to water-soluble disaccharide known as maltose. The net result is the removal of glycogen from the tissue section prior PAS technique. Thus, the staining loss following diastase digestion is indicative of glycogen [Figure 3]b. In our study when we compared the PAS and PAS-D scores in the study population, we found a significant difference in PAS-positive cells in diabetics [Table 1]. Similar results were obtained by Latti et al. This proves that PAS positivity in diabetics is because of increased intracellular glycogen accumulation.
|Figure 3: (a) PAS-positive cells (10×). (b) Decreased PAS positivity following Diastase digestion thus confirming the presence of intracellular glycogen (10×)|
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To know if there is increased intracellular glycogen accumulation in diabetes, we compared the number of glycogen-containing cells in healthy individuals with diabetics and found significantly increased number of glycogen containing cells in diabetic individuals than healthy individuals [Table 2]. Similar results were obtained by Latti et al., Satpathy et al., Ravindran et al., Hallikerimath et al., Asemi-Rad et al., and Reddy et al. This may be because of the increased transport of glucose into the cell through GLUT-1 secondary to the increased blood-glucose level in diabetics.
Having found that there is increased intracellular glycogen accumulation in diabetes, we wanted to know if the duration of diabetes has any role in intracellular accumulation, so we compared the number of PAS-positive cells (score 1 and 2) in case groups but we did not find any significant difference [Table 3]. So, the intracellular glycogen accumulation might depend on various factors such as glycaemic control rather than the duration of diabetes.
To know if we can evaluate the blood-glucose level based on PAS staining, we tried to find the correlation between RBG and the number of PAS-positive cells but found no significant correlation [Table 4]. A similar result was obtained by Ravindran et al. and Fasske et al., This is in contrast with the result obtained by Yasminsatpathy et al. and Reddy et al.
The lack of correlation could be because intracellular glycogen accumulation depends on blood-glucose level over a period of time and also on the turnover rate of the cell. On the other hand, blood-glucose levels are in a constant state of fluctuation depending on the medication status and food intake.
From our observation, we can say that PAS staining helps to convey the glycaemic status of an individual in the recent past and not on a real-time basis. The added advantage of this is that such noninvasive method to assess the glycaemic status will be a boon, especially for patients with compromised medical conditions limiting venipuncture such as hemophilia and debilitated or older individuals with poor venous access who require frequent monitoring of their health status.
The other observations in our study were increased binucleation, presence of micronuclei, presence of more microbial colonies, fungal colonies, and also yeast cells [Figure 4]. These findings were more pronounced in case group II that is in a longer duration of diabetes than case group I and control individuals.
|Figure 4: Other observations: (a) Binucleation (PAS, 40×)). (b) Yeast (PAS, 10×). (c) microbial colonies (PAS-D, 40×) (d) Micronuclei (PAS, 40×). (e) Fungal colonies (PAS, 40×)|
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| Conclusion|| |
From the study, we draw the following conclusions: (1) PAS positivity is due to intracellular glycogen accumulation in type-2 diabetics and (2) the PAS staining can only convey the glycaemic status of an individual in the recent past instead of real-time values. In other words, we can state that PAS staining of exfoliated cells of buccal mucosa will have a better role in screening and therapeutic monitoring noninvasively and to that extent adds value to the body of the research. Promising results of our study could be taken up on a larger scale for gaining more insights in future research.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Wild S, Roglic G, Green A, Sicree R, King H. Global prevalence of diabetes: Estimates for the year 2000 and projections for 2030. Diabetes Care 2004;27:1047-53.
Olefsky JM, Revers RR, Prince M, Henry RR, Garvey WT, Scarlett JA, et al
. Insulin resistance in non-insulin dependent (type II) and insulin-dependent (type I) diabetes mellitus. Adv Exp Med Biol 1985;189:176-205
Wu Y, Ding Y, Tanaka Y, Zhang W. Risk factors contributing to type 2 diabetes and recent advances in the treatment and prevention. Int J Med Sci 2014;11:1185.
Berg JM, Tymoczko JL, Gatto GJ Jr. Stryer: Biochemistry. Vol 5. WH Freeman and Company; 2002. p. 306-7.
Latti BR, Birajdar SB, Latti RG. Periodic acid Schiff-Diastase as a key in exfoliative cytology in diabetics: A pilot study. J Oral Maxillofac Pathol 2015;19:188.
] [Full text]
Shafiei MT, Gonczi CM, Rahman MS, East A, François J, Darlington PJ. Detecting glycogen in peripheral blood mononuclear cells with periodic acid schiff staining. J Vis Exp 2014;94:1-8.
Sahu M, Suryawanshi H, Nayak S, Kumar P. Cytomorphometric analysis of gingival epithelium and buccal mucosa cells in type 2 diabetes mellitus patients. J Oral Maxillofac Pathol 2017;21:224.
] [Full text]
American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care 2014;37(Suppl 1):S81-90.
Roach PJ, Depaoli-Roach AA, Hurley TD, Tagliabracci VS. Glycogen and its metabolism: Some new developments and old themes. Biochem J 2012;441:763-87.
Straseski JA, Gibson AL, Thomas-Virnig CL, Allen-Hoffmann BL. Oxygen deprivation inhibits basal keratinocyte proliferation in a model of human skin and induces regio-specific changes in the distribution of epidermal adherens junction proteins, aquaporin-3, and glycogen. Wound Repair Regen 2009;17:606-16.
Aizawa H, Yamada SI, Xiao T, Shimane T, Hayashi K, Qi F, et al.
Difference in glycogen metabolism (glycogen synthesis and glycolysis) between normal and dysplastic/malignant oral epithelium. Arch Oral Biol 2017;83:340-7.
Satpathy Y, Kumar PS, Singh N. Promising role of exfoliative cytology in the evaluation of glycaemic status of type II diabetics: A pilot study. J Oral Maxillofac Surg 2015;14:206-11.
Ravindran R, Gopinathan DM, Sukumaran S. Estimation of salivary glucose and glycogen content in exfoliated buccal mucosal cells of patients with type II diabetes mellitus. J Clin Diagn Res 2015;9:ZC89.
Hallikerimath S, Sapra G, Kale A, Malur PR. Cytomorphometric analysis and assessment of periodic acid schiff positivity of exfoliated cells from apparently normal buccal mucosa of type 2 diabetic patients. Actacytologica 2011;55:197-202.
Asemi-Rad A, Heidari Z, Mahmoudzadeh-Sagheb H. The relationship between the staining intensity of oral exfoliative cells with periodic acid schiff and cytomorphometric indices with fasting blood sugar in type 2 diabetic patients. Interdiscip J contemp Res Bus 2013;4:375-81.
Reddy M, Manyam R, Swetha P. Comparison of glycogen positive cells in oral smears with random blood sugar levels of type 2 diabetes patients. Ann Med Health Sci Res 2018;8:1-5.
Falin LI. Glycogen in the epithelium of mucous membranes and skin and its significance. Acta Anat (Basel) 1961;46:244-76.
Fasske E, Morgenroth K. Comparitive stomatoscopic and histochemical studies of the marginal gingiva in man. Parodontologie 1958;12:151-60.
Prasad H, Ramesh V, Balamurali PD. Morphologic and cytomorphometric analysis of exfoliated buccal mucosal cells in diabetes patients. J Cytol 2010;27:113.
] [Full text]
Dr. S Shamala Ravikumar
Department of Oral Pathology, Adhiparasakthi Dental College and Hospital, Melmaruvathur, Tamil Nadu
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4]