The study evaluated the effect of different doses of metformin on the intermediates of NO biosynthesis in people with pre-diabetes.
Patients were recruited from Diabetes Centre between October 2017 and December 2018, after the agreement (no: ST.C310.17.009) with Wroclaw Medical University Bioethical Committee was concluded, and patients’ written consent was obtained. The full trial protocol, the flow-chart of the patients, as well as detailed inclusion and exclusion criteria can be accessed at ClinicalTrials.gov Registration webpage.
The metformin-naïve patients (age: 40–65 yo) with new-diagnosed pre-diabetes and without previously diagnosed cardiovascular diseases, were treated during three weeks with an increasing dose of the immediate-release (regular form) metformin to 3 × 500 mg, if no contraindication was present, and then randomly assigned to: group A – continuation of a dose of 3 × 500 mg or group B – dose increased to: 3 × 1000 mg (also by titration). After 12 weeks of the different dose treatment mentioned above, group B returned to the dose of 3 × 500 mg for the next three weeks. Thus the total treatment period lasted 15 weeks with a fixed dose of 3 × 500 mg for group A, and variable dose (with max dosage of 3 × 1000 mg) for group B. During titration period (by phone) and blood collection visits (personally), patients were asked about the compliance. They were also encouraged to contact in any case of the problem with metformin tolerance. If potential side effects appeared in group B, the patients could take a lower dose (3 × 500 mg, if well-tolerated) thus, being assigned to group A. If the smallest dosage (3 × 500 mg) was not well tolerated, the patient was excluded from the study.
The statistician and the team, responsible for biochemical and statistical analysis, had no information on the dose administered to patients (single-blinded study). The nurse randomised patients regarding to the identification number (ID): even or odd second digit, which is personal and individual for each inhabitant in the country.
Pre-diabetes was defined as impaired fasting glucose (IFG) or/and impaired glucose tolerance (IGT) according to the local, as well as the European Association’s for the Study of Diabetes (EASD) criteria (24). The results of fasting glucose (FG) and oral glucose tolerance test (OGTT) had been obtained before the patient from treatment groups started the study.
Before the administration of the first dose of the metformin and after: 6, 12, 15 weeks of the treatment, the blood concentration of metformin, as well as the compounds related to the production of nitric oxide were assessed for group A and B. The wide panel of L-arginine/NO pathway metabolites (L-arginine, L-citrulline, ADMA – asymmetric dimethylarginine, DMA – dimethylamine, SDMA – symmetric dimethylarginine, ornithine) were assessed by a recently developed novel assay (25), using the liquid chromatography-mass spectrometry technique (LC-MS/MS).
The admissible delay in taking blood for lab-testing was set at four days. The blood samples were taken randomly (both: time and gaps between metformin administration were not defined).
The blood samples from matched healthy volunteers (group C) were taken once to compare the initial parameters as this group was not treated with metformin.
The basic parameters for all three groups (A,B,C) like: lipid profile, HbA1c etc. (Table no1) were obtained from patients’ medical records and considered relevant, if they had been taken, up to six months earlier.
The specialistic parameters like metformin concentration or concentration of the compounds related to the production of nitric oxide were assessed in the laboratory of the Department of Medical Biochemistry at Wroclaw Medical University.
The Statistica 13 programme was used with cut-off point for statistical significance (P) at 0.05. In order to determine the significance, statistical tests were used in accordance with the distribution of variables and the nature of the data (Student t test, Welch’s test, Mann-Whitney U test, chi – square test, the Wilcoxon signed-rank test, Kruskal – Wallis test, Friedman test, F test).
The funding source had no involvement in any part of the study conduction. The corresponding author confirms that had full access to all the data in the study and assumes final responsibility for the decision to submit it for publication.