Use of novel biomarkers (Homocysteine, vitamin B6, B9 and B12) on the assessing the progression of cardiovascular disease

A large number of evidences correlate elevated levels of homocysteine (Hcys) with a higher cardiovascular diseases (CVDs) risk, especially, atherosclerosis. Similarly, abnormal low levels of the vitamins B6, B9 and B12 are associated to an instability in the methionine cycle with an over production of Hcys. Thus, biomedical sciences are looking forward for a cheaper, faster, precise and accurate analytical methodology to quantify these compounds in a suitable format for the clinical environment. Therefore the objective of this study was the development of a simple, inexpensive and appropriate methodology to use at the clinical level. To achieve this goal, a procedure integrating a digitally controlled (eVol®) microextraction by packed sorbent (MEPS) and an ultra performance liquid chromatography (UPLC) coupled to a photodiode array detector (PDA) was developed to identify and quantify Hcys vitamins B6, B9 and B12. Although different conditions were assayed, we were not able to combine Hcys with the vitamins in the same analytical procedure, and so we proceeded to the optimization of two methods differing only in the composition of the gradient of the mobile phase and the injected volume. It was found that MEPS did not bring any benefit to the quantification of the Hcys in the plasma. Therefore, we developed and validate an alternative method that uses the direct injection of treated plasma (reduced and precipitated). This same method was evaluated in terms of selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), matrix effect and precision (intra-and inter-day) and applied to the determination of Hcys in a group composed by patients presenting augmented CVD risk. Good results in terms of selectivity and linearity (R2> 0.9968) were obtained, being the values of LOD and LOQ 0.007 and 0.21 mol / L, respectively. The intra-day precision (1.23-3.32%), inter-day precision (5.43-6.99%) and the recovery rate (82.5 to 93.1%) of this method were satisfactory. The matrix effect (>120%) was, however, higher than we were waiting for. Using this methodology it was possible to determine the amount of Hcys in real plasma samples from individuals presenting augmented CVD risk. Regarding the methodology developed for vitamins, despite the optimization of the extraction technique and the chromatographic conditions, it was found that the levels usually present in plasma are far below the sensitivity we obtained. Therefore, further optimizations of the methodology developed are needed. As conclusion, part of the objectives of this study was achieved with the development of a quick, simple and cheaper method for the quantification of Hcys.