Methylglyoxal Could Mediate the Relationship Between Gum Infections and Diabetes: A Method Development and Feasibility Study
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Background: MGO is an α-dicarbonyl compound that is present in all the cells, under both normal and pathological conditions. It is a toxic by-product of glycolysis in humans and is produced in high levels in other living organisms, particularly certain oral bacteria. The level of MGO in the body has been shown to be associated with diabetes and periodontal infections. The goal of this project is to develop and validate a Liquid Chromatography based technique for quantification of free MGO and compare these results with the widely used ELISA assay for quantification of protein bound MGO in human serum. Methods: Three diamino compounds were investigated for the ability to form quantitative derivatives suitable for high performance liquid chromatography (HPLC) separation and fluorescence (FL) or mass spectrometry (MS) detection. The selected derivatization scheme was then validated for linearity, accuracy and precision and applied to the analysis of cultures of Tannerella forsythia ( a periodontal pathogen known to secrete MGO), and to free MGO in 76 human serum samples from participants with and without known diabetes and with and without clinically documented periodontal disease. Simultaneous analyses of bound MGO were made using an ELISA kit from Hycult BioTech. Correlation between methods was assessed by linear regression. Differences between disease conditions was assessed by ANOVA. Results: Reverse-phase LC using Acentis C18 column with a binary gradient using Acetonitrile and water performed with derivatization of MGO with 1,2 diamino, 4,5 dimethoxy benzene (DMB) provided the best product for separation and MS detection. MS detection using ESI interface and selective ion monitoring yielded limits of detection of 1ng/mL for MGO. Stability studies indicated MGO to be stable for 2 freeze-thaw cycles but the compound formation was observed during the third freeze-thaw cycle. Estimate of imprecision in BSA and normal human serum sample were < 15% for MGO. Analysis of culture supernatants from the wild-type and a deletion mutant of T. forsythia lacking the mgsA (methylglyoxal synthase) gene showed the ability of methylglyoxal synthase to increase the production of MGO in organism. A pilot study with human serum samples (N= 76) belonging to four different classes of disease conditions namely, D+ P+, D-P+, D+P- and D-P- were analyzed by both LC-MS assay and ELISA. Effect of BMI, age and sex of the patients when compared with their endogenous MGO levels, showed no direct relationship. However, mean MGO in men were found to be higher when compared to women, although these differences were not statistically significant. Differences in mean MGO between the four different clinical groups defined by diabetes and periodontal disease status were not statistically significant. Conclusion: A new LC-MS method with DMB derivatization was developed for analysis of MGO in human serum samples. Application of this assay on wild type and mutant T.forsythia cultures showed the ability of methyglyoxal synthase to contribute to the overall MGO production by the organism. Pilot study with biorepository serum samples from patients with diabetes and periodontal infection analyzed by LC-MS and ELISA assay produced results that showed no clear relationship between MGO and diabetes or periodontal infections in vivo. Further consideration of certain factors such as severity of the diseases and smoking status of patients, and how they potential influence MGO concentrations in relation to patient’s clinical status could provide helpful insight to clarify the present results and should be a focus in future investigations.