Assessing cardiotonic steroids involvement in hypertensive rat models with Helicobacter pylori infections

Abstract

Introduction: Hypertension is an important public health challenge worldwide, being the leading cause of cardiovascular disease, morbidity and mortality. It is particularly prevalent in people in sub-Saharan Africa, especially in urban areas. There is an urgent need to develop strategies to prevent, detect, treat, and control hypertension effectively in the African region. Helicobacter pylori, a gram-negative bacterium responsible for many gastric disorders worldwide, has been associated with hypertension in some previous studies; where blood pressure of patients with Helicobacter pylori infection did not subside after hypertensive treatment, when compared to patients without Helicobacter pylori infections. This effect was suggested to be due to Helicobacter pylori produced and modified cardiotonic steroids that are found in elevated concentrations in hypertensive patients. Cardiotonic steroids are positive inotropic agents which are known to increase blood pressure. A sensitive analytical method is needed to detect and quantify the low concentrations of cardiotonic steroids in biological samples. Materials and Methods: An extraction method was optimised using reversed phase Solid Phase Extraction. A targeted liquid chromatography tandem mass spectrometry method using an Agilent binary series 1100/1200 LC system with a Kinetex C18 RP column (100 x 2.1 mm, 2.6 µm) coupled to a Sciex 4000QTRAP tandem mass spectrometer was developed and validated for the detection and quantitation of 9 different cardiotonic steroids in both solvent and whole blood. The method was validated according to the International Conference on Harmonization guidelines with regards to precision, accuracy, sensitivity, selectivity, linearity, range, limit of detection, limit of quantification, reproducibility, recovery, carry-over and stability. Media from Helicobacter pylori cultures and faecal samples from human and different normo- and hypertensive rat strains were analysed. Data analysis was performed with Analyst® Software (version 1.5.2) and multiple t-test and Kruskal Wallis test using GraphPad Prism 8 software.

Results and Discussion: The calibration curves of tested cardiotonic steroids were linear over a concentration range of 0.1-40 ng/mL with coefficients of determination greater than 0.990 except for telocinobufagin. The analytical method was selective with an estimated limit of detection and limit of quantification between 0.02-0.5 ng/mL and 0.1-2 ng/mL respectively. All tested cardiotonic steroids showed good recovery of over 70%. Accuracy and precision were found to be within acceptable limits of 15% and 20% at lowest limit of quantification for almost all the analytes and their stability in blood and solvent at room temperature, 4°C, -20°C and -80°C was tested for a month. Cardiotonic steroids were detected in Helicobacter pylori cultures and faecal samples with the exception of ouabain and proscillaridin A which were not detected at all. Although Helicobacter pylori were shown to produce cardiotonic steroids in vitro, no evidence of the effect of Helicobacter pylori on cardiotonic steroids production was detected in different normo- and hypertensive rat groups. Conclusion: The quantitative analytical method was successfully validated, over expected in vivo concentration ranges for 8 different cardiotonic steroids. The extraction and analytical methods were both successfully applied to Helicobacter pylori cultures and faecal rat samples where cardiotonic steroids were detected.