EX-VIVO STUDY TO DETERMINE THE EFFICACY OF A FORMULATION IN ELIMINATING EXCESS SALIVARY GLUCOSE IN DIABETIC SUBJECTS
Mme Françoise Bafort – Liège University – Agro-Biotech-Passage des Déportés 2 – 5030 Gembloux – Belgium. Presented at the Opening Symposium of the Brussels Diabetes Research Pole, Free University of Brussels, 26th February 2016
There are a number of important oral manifestations of diabetes. Dental caries is one of the most common oral diseases which is modified when diabetes is present. Root caries is more prevalent in patients with diabetes. In this study, we have demonstrated that it is possible to eliminate the excess of glucose contained in the saliva of Diabetics. That could be one of the solutions to avoid infection, pain, tooth loss and reduce masticatory functions due to dental caries.
There are a large range of oral manifestations that have been reported in patients with diabetes. These include increased extent and severity of periodontal disease, changes in the prevalence of dental caries, burning mouth syndrome, Candida infection, Xerostomia (Dry mouth), altered taste sensation, altered tooth eruption, and hypertrophy of the parotid glands (Park et al., 2009). Non-dental health care providers and patients need to be aware of the changes in the oral cavity associated with diabetes, emphasize the importance of an oral evaluation when the diagnosis of diabetes is first made, and make appropriate referral if the patient reports a problem. Dental professionals have to be familiar with the range of oral disorders in patients with diabetes, and how these problems should be managed in patients with the disease. The results of the ex-vivo tests described in this paper, explained how one of the two common oral diseases (periodontal and caries) might be reduced. The method comprises the use of oral health care products formulated with a “Milk Protein Extract” which has been shown to decrease salivary glucose in the diabetic mouth which could lead to a control of the caries.
The hexokinase method which has been used comprises analyzing the concentration of glucose in a solution or in the saliva of diabetic subjects before and after the use of a Milk Protein Extract (MPE) which is one of the active ingredients of new oral care formulations called AnOxident balance. The determination of the glucose concentration in saliva or any other solutions is carried out by the use of the hexokinase enzyme by the following reaction:
hexokinase Glucose + ATP → glucose-6-phosphate (glucose-6-P) + ADP G6PD (*) Glucose-6-P + NADP+ → 6-phospho-gluconolactone + NADPH + H+
(*) G6PD: glucose-6-phosphodehydrogenase
Glucose standard solution
A mother solution of 1000µM glucose was prepared (standard solution). Different dilutions of the standard solution of glucose were prepared by diluting 10 times with a buffer containing 100 mM HEPES, MgCl2 12 mM, KCl 120 mM and KH2PO4 20 mM – at pH 7.5 adjusted with NaOH 1M. The different glucose solutions having a final concentration between 5 and 500 µM was analyzed using the following method:
100 µl glucose solution was mixed with 95 µL of reagent medium containing 2.0 mM MgCl2, 0.5 mM ATP, 0.5 mM NADP+, and 0.06 units of yeast glucose-6-phosphate dehydrogenase in TRIS-HCL buffer (200 mM, pH 8.1). After a reading of the absorbance at 340 nm, the reaction was started by the addition of 5µl hexokinase in reagent medium (0.06 units). The absorbance was read each minute over a 5-minute period at room temperature. After adding the hexokinase, the solutions were mixed by inversion of the cuvets. The absorbance was recorded after 30-minutes incubation at room temperature. The assay was simultaneously conducted on different glucose solution prepared from the standard solution (final concentration comprised between 5 and 500 µM).
The dosage method is a method in final point. The height of the plateau is proportional to the glucose concentration.
The results were calculated as µmole of glucose/l after the subtraction of reading the absorbance of the control (solution which does not contain the hexokinase). The standard curve of glucose between 5 to 500 µM is linear and is represented in the figure 1.
Figure 1: Standard curve of the determination of the glucose concentration in function of the of the NADPH production using the hexokinase method
Milk Protein Extract (MPE)
The MPE is a complex of proteins which is able to transform, in presence of ATP, the α-D-glucose to α-D-glucose-6-phosphate and ADP. It catalyzes the transformation of the α-D-glucose as follows:
MPE D-Glucose + ATP (*) → α-D-Glucose-6-phospahte + ADP
(*) ATP: Adenosine 5’-triphosphate, presented in the MPE comes from the calcium phosphate-citrate (CPC)-caseinate micelles contained in raw milk (Richardson et al., 1980).
The glucose at pH 7 is under a cyclic form having 36.4 % of β-D-glucose and of 63.6 % of α-D-glucose. The linear form represents a negligible fraction
The oxidation of the α-D-glucose changes the equilibrium and β-D-glucose is transformed in α-D-glucose, as follows:
α-D-glucose ↔ β-D-glucose
The action of the MPE on the α-D-glucose transforms this ingredient into α-D-glucose-6-phosphate. The reaction can be measured by the increase of the NADPH in the presence of NADP+ and the α-D-Glucose-6-phosphodehydrogenanse. In our experiments, we have analyzed the concentration of the glucose in the solution by the “hexokinase” method.
Evaluation of the glucose concentration after its reaction with the MPE
After incubation at 37°C during variable time until 120 seconds, 1 ml of the solution containing 500 µM of glucose and 2 mg of MPE is immersed in a bain-marie at 80°C (temperature of the denaturation of the MPE proteins) to stop the reaction.
– 100 µl of this solution is taken to dose the concentration of glucose which is still remained after the action of the MPE by the method of the hexokinase as described in the paragraph “Hexokinase”. The results are described in the Figure 2
Saliva sample collection
To collect saliva, a standardized tube with two compartments and a standardized cotton were used. The upper part of the tube containing the cotton presented a hole, so that, after centrifugation, the saliva was recorded in the lower part and became available for analysis.Saliva was collected from the subjects, immediately after rinsing the oral cavity twice with 150 ml of water and drinking this water, by means of cotton kept in the oral cavity for 2 minutes either in the unstimulated state or during mastication (stimulated saliva). The cotton was transferred into the upper part of the tube. Salivary flow was determined by weighing the device with the cotton before and after saliva collection, assuming that 1 g of saliva correspond to 1 ml. Centrifugation of the device at 2000 g for 5 minutes allowed saliva adsorbed to the cotton to pass through the orifice into the lower compartment of the device. The saliva was split in two parts, one for the control and one treated directly with the MPE
The two compartments tube and the cotton were obtained from the same manufacturer Salivette Starsted, Numbrecht, Germany.
HEPES from Gibco (11344-033), MgCl2 (Merck 5833), KCl (Merck 4935), KH2PO4 (Merck 4873), NADP+, G6PD, ATP were bought from Merck. Hexokinase (180 mU/essay) has been bought from Sigma.
MPE was supplied by Taradon Laboratory. It is a mixture of protein which has been extracted from raw milk using a specific mixed mode resin chromatography hydrophobic – ionic exchange.
A study was conducted previously to determine the specificity of the MPE. It is important to know that the MPE is really specific to the D-glucose and will not transform other glucosides which are contained in the food as follows: lactose, maltose and fructose. It is the same for starch and hydrolyzed starch. We also determined the optimal pH of the activity of the MPE which ranges from 5.5 to 7, which is similar to salivary pH, and the optimal temperature which is between 37°C to 40°C which is also similar to the temperature of saliva.
Furthermore, we analyzed the degradation of the standard glucose solution (500 µm/l) by the MPE. As it is shown in the figure 2, in less than 2 minutes, it is possible to eliminate all glucose contained in a solution of 500 µM glucose.
Figure 2: Elimination of a standard glucose solution (500 µM/l) by the MPE
The second part of the study was conducted in 6 diabetic subjects and 3 normal subjects. In normal subjects, the glucose concentration was on average 68.4 µM in unstimulated saliva and an average of 45.3µM in stimulated saliva. In the case of the diabetic subjects, we found a glucose concentration of an average of 264 µM in unstimulated saliva and an average of 255 µM in stimulated saliva as it is summarized in the table 1.
The present results confirm that the glucose concentration in saliva is higher in diabetic patients that in normal subjects. It also confirms that, both for normal subjects and diabetic subjects, the saliva flow is higher in stimulated as compared to unstimulated saliva.
The study was carried out to check if the MPE contained in the AnOxident balance formulations, is able to decrease the salivary glucose quantity. In fact, when we collected the saliva, we treated the sample with the MPE formulation and we stopped the reaction at 30 seconds, 60 seconds and 120 seconds. Using the methodology of the “hexokinase” method, we analyzed the concentration of the glucose which remained in the saliva samples after the treatment by the MPE. The results are summarized in the table 1:
Normal Diabetic Glucose concentration (normal) Glucose concentration (diabetic) 30 sec. 60 sec. 120 sec. 30 sec. 60 sec. 120 sec. Glucose concentration (µM) µmole/l µmole/l µmole/l µmole/l µmole/l µmole/l Unstimulated 68.4 +/- 7.4 264 +/- 10.5 Stimulated 34.3 +/- 8.6 255 +/- 11.3 Salivary flow (ml/min) Unstimulated 0.96 +/- 0.11 1.27 +/- 0.12 Stimulated 1.92 +/- 0.12 1.48 +/- 0.11 Glucose excretion (nmol/2min) Unstimulated 134.2 +/- 6.8 670.8 +/- 0.12 67 53.58 40.26 254.904 127.452 67.08 Stimulated 131.6 +/- 13 755.4 +/- 0.11 65.8 52.64 39.48 287.052 143.525 75.54
Table 1: Decrease of the salivary glucose in normal and diabetic subjects using MPE after 30, 60 and 120 seconds’ contact
As we can observe, in the case of a standard glucose solution, we can eliminate almost all the quantity of glucose due to the presence of the MPE. On the other hand, this degradation of the excess of glucose is not total when the tests are carried out ex-vivo with saliva. Visibly, a certain quantity of the salivary glucose stays more or less constant in the saliva after the application of the MPE. That has to be due to an inhibitory effect due to the presence of other molecules in the saliva. Nevertheless, it is possible to eliminate the excess of glucose reaching a concentration similar to the level of the one existing in the normal subjects.
Discussion and Conclusions
Several studies based on clinical observation have suggested that the teeth of diabetic patients are predisposed to dental caries (Miralles et al, 2006 – Taylor et al., 2004 – Twetman et al., 2002). Other studies on animals reported the same (Borghelli et al., 1966 – Hartles et al., 1958). Dental caries is the indirect results of the metabolism of fermentable carbohydrates by specific oral bacteria such as Streptococcus mutans, Lactobacillus species, with the by-product of the metabolism which is lactic acid acting on a mineralized substrate which is the dentition. The rate of D-glucose utilization by oral bacteria at a physiological concentration of D-glucose in saliva (50 µM) was estimated at 0.047nmole/min per 106 bacteria (Cetik et al., 2013). This demonstrates that the glucose concentration in stimulated and unstimulated saliva in diabetics is important enough for the bacteria to metabolize the lactic acid. The result is the demineralization of the teeth, ultimately leading to cavities. Advanced dental caries is associated with involvement of the neurovascular tissue within pulp chamber of the teeth, resulting in pain and abscess formation that lead to the tooth extraction.
In this work, we have demonstrated ex-vivo the possibility to eliminate the excess glucose contained in the diabetic saliva. Using a specific formulation called “Milk Protein Extract”, it is possible to eliminate the glucose in excess glucose in the diabetic saliva reaching a level similar to that one existing in the saliva of normal subjects. Although some scientists are not convinced that there is a relation between diabetes and the unusual appearance of dental caries, we think that we cannot exclude the fact that a constant production of saliva during the day, having an excessive concentration of glucose (4 to 5 times the concentration of glucose in normal subject) is a factor causing caries. On the other hand, considering the pathogenesis of dental caries, it should be noted that the lesions have a multifactorial etiology involving 4 main factors: glucose and other sugars in food, acid producing pathogenic bacteria, predisposing host factors such as behavioural characteristics of the host that affect susceptibility to dental caries) and time (Kyes et al., 1961 – Reich et al., 1999).
Nevertheless, we cannot consider that the elimination of the excess glucose is going to solve all the problems of oral health in diabetics. As we have underlined previously, the other common oral complication of diabetes is periodontal disease. Periodontal diseases are a group of inflammatory disorders of the supporting tissues of the teeth, which includes the gingiva (mucosal tissue around the teeth, cementum, the periodontal ligament). The pathology of the disease is an inflammatory lesion induced by the biofilm bacteria, damaging the gum cells and provoking the oxidative stress which corresponds to an over-excess of the free radical production versus the antioxidant activity molecules. The composition of the MPE comprises several ingredients such as the vitamins E and C, glycine, glutamic acid and cystine as precursors of glutathione and also glutathione itself. The formulation also contains the DUOX system which is able to control the H2O2 produced by the free radicals in inhibiting the growth of the bacteria in presence of the excess of glucose.
In the final formulation of AnOxident balance, in addition to MPE, there is anti-oxidant plant extract which neutralizes the oxygen radical (O2°-) and apo-lactoferrin which chelates iron, to avoid the production of the hydroxy free radical (OH°). All these elements play an important role in neutralizing the free radicals responsible for the inflammatory problems of the gum cells (gingivitis). MPE also contains certain elements susceptible to controlling the growth of the microorganisms that are organized in biofilm or in planktonic forms, which could impact on repair of the mucosal cells damaged by the bacterial lipopolysaccharides.
It would be interesting to carry out other in vivo studies on diabetic subjects to assess the activity of the complete formulation of the AnOxident balance product to see how the combination of ingredients acting at different levels in the mouth, can bring a general improvement in the oral health of the diabetic subjects.
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