Background and aim: Minimal hepatic encephalopathy (MHE) is the earliest form of hepatic encephalopathy (HE) and can affect up to 80% of patients with liver cirrhosis. It is characterized by impaired cognitive function; mainly in the domains of attention, memory, speed of response, surveillance and integrative function. Patients with MHE show reduced performance in selective and which has a negative impact on the patients’ health-related quality of life. Currently, there is no gold standard for diagnosis of EHM. Reports Montoluiu et al. evaluated the serum levels of different nitro-oxidative stress metabolites, cyclic guanosine monophosphate (cGMP), nitrites+nitrates and 3-nitrotyrosine (3-NO-Tyr); For each metabolite, its diagnostic precision was evaluated as an indicator of MHE, correlating it with the level of performance in psychometric tests for the diagnosis of HE as a comparison, finding high sensitivity and specificity for 3-nitrotyrosine. Despite the fact that 3-NO-Tyr has been evaluated in EHM, there is a wide variation of results that support its clinical utility, mainly due to the quantification methods used. AIM. To develop an analytical method to quantify the products of nitro-oxidative stress 3-NO-Tyr and 3-chloro-Tyrosine (3-Cl-Tyr) of high sensitivity and specificity to quantify the levels of these metabolites in samples from participating subjects with liver damage and MHE and comparing against the levels of subjects with liver damage without MHE, as well as the baseline level in healthy control subjects.
Material and methods: This study was approved by the Institute's Ethics and Research Committee. An analytical method was developed for the quantification of 3-NO-Tyr as 3-Cl-Tyr by means of triple quadrupole mass spectrometry coupled to an ultra high efficiency liquid chromatography system (UPLC-MS / MS XEVO TqD Waters). The spectrometer was programmed using the molecular transitions of NO-Tyr (227.2> 181.1), Cl-Tyr (216.2> 170.1) and the internal standard (EI) (132.2>86.2) for the internal standard respectively. The samples were hydrolyzed prior to processing and analysis to quantify free and protein-derived metabolites.
Results: The method was linear in the range of 0.5 - 2500nM for both metabolites, it met the validation tests of the analytical methods. The results show that, by means of the developed method, it is possible to perform the simultaneous quantification of free 3-NO-Tyr and Cl-Tyr and protein derivatives, so it can be used to quantify them in samples of MHE, non-MHE and control subjects.
Conclusions: With the developed method, it is possible to accurately and precisely quantify the concentrations of both metabolites in the proposed biological matrix, so if they show differences between study groups, they could be used to determine them in the early diagnosis of MHE.
Financing: Project supported by the SS / IMSS / ISSSTE-CONACYT Fondo sectorial de investigación en salud 1-2017. 289979. Project supported by Program E015 Research and Technological Development in Health of the Instituto de Seguridad y Servicios Sociales de los trabajadores del Estado.
Conflicts of interest: The authors have no conflicts of interest to declare.
