A SLICE OF CHEESE ON SIR ANDREW AGUECHEEK’S TABLE

oil painting by Joseph William Turner

THE SIMULTANEOUS QUANTIFICATION OF BIOMARKERS OF OXIDATIVE STRESS IN THE DIAGNOSIS OF MINIMAL HEPATIC ENCEPHALOPATHY

Who would have thought that the spectacular sunsets painted by Joseph Mallord William Turner in the Romantic period were created during the famine provoked by the eruption of the volcano Mount Tambora in 1815?

And what does this have to do with William Shakespeare’s play Twelfth Night, written in 1605? In Act 3, Scene 1, Sir Andrew Aguecheek, a silly old ale-loving squire, declares: “I am a great eater of beef, and I believe that does harm to my wit.” Shakespeare is presenting a classic example of a case of hepatic encephalopathy caused by intolerance to proteins (Guillén et al., 2003). He is illustrating, moreover, the complex suffering of human beings in the face of chronic liver disease (CLD): the ravages of cirrhosis on the social cognition of those suffering from this condition and their family members (Bajaj, 2008).

Hepatic encephalopathy (HE) is a neuropsychiatric and cognitive disorder caused by decompensated cirrhosis (Nardone et al., 2016). Also called “manifest” HE, this condition is a frequent complication among cirrhosis patients, affecting both quality of life and mortality rates. Minimal hepatic encephalopathy (MHE) is found in at least half of cirrhosis patients (Maldonado-Garza et al. 2011) and increases the risk of progression to HE (Kaccaamy and Bajaj, 2011; Labenz et al., 2018). MHE is a condition characterized by subtle (and reversible) anomalies that can easily pass unnoticed during a medical examination. Using the SONIC model (Spectrum of Neurocognitive Impairment in Cirrhosis), Bajaj has described the neurological deterioration as a natural consequence of chronic liver disease (Bajaj et al., 2011). Staring from a normal neurological state, the patient will undergo gradual neuronal degeneration, eventually ending in a state of coma and death due to CLD.

In order to prevent the development of HE, early intervention at the stage of MHE, based on a precise diagnosis, is fundamental. Patients with MHE show a reduction in the performance of daily activities, making for a shorter productive life, with attention deficits, memory gaps, slowed responses, and reduced visual-motor integration. There is no gold standard currently for the diagnosis of MHE (Maldonado-Garza et al. 2011), but the condition can be detected by a combination of specific neuropsychometric and/or neurophysiological tests for patients with CLD (Ridola et al., 2018), although this requires a large investment of clinical practice time, since the tests need to be adapted to the age and education level of the patient, and both skilled personnel and expensive equipment are necessary to administer them. As a result, most cases of MHE go undiagnosed and fail to receive timely treatment, with a direct impact on the quality of life and life expectancy of patients with cirrhosis.

Certain metabolic alterations have been identified in the brains of MHE patients. Montoliu and colleagues evaluated the serum levels of various nitro-oxidative stress metabolites, the cyclic guanosine monophosphate, nitrites + nitrates, and 3-nitrotyrosine (3-NO-Tyr). Although 3-NO-Tyr has not been evaluated in HME, general results to measure its clinical utility have shown wide variations. As a result, it has not been recommended for the purposes of diagnosis.

3-NO-Tyr is a product of tyrosine nitration. The word “tyrosine” comes from the Greek word tyros, meaning “cheese,” and was discovered in 1846 by the German chemist Justus von Liebig. At the age of thirteen, Liebig had been inspired by those very same sunsets captured by Turner during the deadliest volcanic eruption in history, which left Europe without a summer in the year 1816. The ruined harvests of that year motivated Liebig to make some important contributions to nutrition, including his special soluble food for babies. Thanks to some of Liebig’s countless contributions to science ―the description of the casein protein in cheese and most of the metabolic pathways of tyrosine―, it has since been found that levels of 3-NO-Tyr in the blood increase significantly among cirrhosis patients with MHE, compared to patients without MHE. The mechanisms whereby this change in 3-NO-Tyr levels occurs in patients with MHE are not currently understood. In order to measure 3-NO-Tyr, the technique of high performance liquid chromatography (HPLC) is used to separate it and then identify it by means of fluorescent light. It can also be located with an electrochemical detector once it has been separated by means of HPLC or other procedures. Nevertheless, the wide variations in the parameters and methods for measuring 3-NO-Tyr (gas chromatography and mass spectrometry), including the ELISA test, using specific antibodies against it, have diminished its diagnostic usefulness.

The oxidation of tyrosine with chlorine can occur through electrophilic aromatic substitution and N-chlorination. In the course of the damage to the liver caused by the infectious process, the tyrosine is degraded into 3-cholotyrosine (3-ClTyr). Cl-Tyr has been thoroughly studied as a biomarker for inflammation and the oxidative tissue damage resulting from the myeloperoxidase staining of neutrophils, in cases of chronic inflammatory disease. Specifically, high levels of Cl-Tyr are associated with renal insufficiency, arteriosclerosis, heart attacks, and cystic fibrosis (Crow et al., 2016).

In order to prevent the development of manifest hepatic encephalopathy, early treatment of minimal hepatic encephalopathy, based on precise diagnoses, is essential. We therefore developed an analytical method to measure simultaneously the products of nitro-oxidative stress 3-NO-Tyr and 3-Cl-Tyr with high sensitivity and specificity, in order to quantify the levels of the metabolites in the samples of subjects with liver damage and MHE, as compared to the levels of subjects with liver damage and no MHE, as well as the base level in control subjects. In order to avoid variations in measurement, we took simultaneous readings using triple quadrupole mass spectrometry combined with a system of ultra performance liquid chromatography (UPLCMS/Waters® Xevo® TQD mass spectrometer) (Villaseñor Todd et al., 2020). The spectrometer was programmed using the specific molecular transitions for each metabolite and the use of an internal standard. The samples were hydrolyzed before their processing and analysis, in order to quantify both the free and protein-derived metabolites.

The results have proved promising, since, by means of this method, the amounts of free and protein-derived 3-NO-Tyr and 3-Cl-Tyr can be measured simultaneously, in samples from patients suffering from cirrhosis with MHE, those without MHE, and control subjects.

The concentrations of the two metabolites in the aforementioned biological matrix can be precisely measured. If there are differences between the two study groups, they can be used in the early diagnosis of MHE.

References: - Bajaj, J. S. Minimal hepatic encephalopathy matters in daily life. (2008). World J Gastroenterol. 2008;14(23):3609-15. - Bajaj. J. S., J. Cordoba, K. D. Mullen, P. Amodio, D. L. Shawcross, R. F. Butterworth, et al. (2011). Review article: The design of clinical trials in hepatic encephalopathy–An International Society for Hepatic Encephalopathy and Nitrogen Metabolism (ISHEN) consensus statement. Aliment Pharmacol Ther. 2011;33(7):739–47. - Crow, B.S., J. Quiñones-González, B. G. Pantazides, J. W. Perez, W. R. Winkeljohn, J. W. Garton, et al. (2016). Simultaneous measurement of 3-chlorotyrosine and 3,5-dichlorotyrosine in whole blood, serum and plasma by isotope dilution HPLC-MS-MS. J Anal Toxicol. 2016;40(4):264–71. - Guillén, J. C. Q., I.C. Soria, J. M. G. Montes, M. J. Sáenz, and J. M. H. Gutiérrez. (2003). La encefalopatía hepática: terminología, etiología y tratamiento. 2003;(5):127-34: <https://idus.us.es/handle/11441/23114> - Kachaamy, T, and J. S. Bajaj. (2011). Diet and cognition in chronic liver disease. Curr Opin Gastroenterol. 2011;27(2):174–9. - Labenz, C., J. S. Baron, G. Toenges, J. M. Schattenberg, M. Nagel, M. F. Sprinzl, et al. (2018). Prospective evaluation of the impact of covert hepatic encephalopathy on quality of life and sleep in cirrhotic patients. Aliment Pharmacol Ther. 2018;48(3):313–21. - Maldonado-Garza, H. J., G. Vázquez-Elizondo, J. Obed Gaytán-Torres, Á. Ricardo Flores-Rendón, M. Graciela Cárdenas- Sandoval, and F. Javier Bosques-Padilla. (2011). Prevalence of minimal hepatic encephalopathy in cirrhotic patients. Ann Hepatol. 2011;10:S40–4: <http://dx.doi.org/10.1016/S1665-2681(19)31605-9>

- Nardone, R., A. C. Taylor, Y. Höller, F. Brigo, P. Lochner, and E. Trinka. (2016). Minimal hepatic encephalopathy: A review. Neurosci Res. 2016;111:1–12: <http://dx.doi.org/10.1016/j.neures.2016.04.009> - Ridola, L., V. Cardinale, and O. Riggio. (2018). The burden of minimal hepatic encephalopathy: From diagnosis to therapeutic strategies. Ann Gastroenterol. 2018;31(2):151–64. - Villaseñor Todd, A., J. A. Hernández-Hernández, R. C. López-Sanchez, F. J. Bosques-Padilla, C. A. Cortez- Hernandez, and E. Lopez Soriano. (2020). Development of the analytical method for the quantification of 3-nitrotirosin and 3-chlorothyrosin in human plasma as potential biomarkers to evaluate minimal liver encephalopathy (MHE). Ann Hepatol. 2020;19(2020):21.

Dra. Ana Villaseñor-Todd

Mexican scientist noted for her research studies in minimal encephalopathy, oxidative stress, quality of life and social cognition. He complemented his graduate studies at Texas A&M University. Candidate to receive the degree of doctor of medicine by the UANL. Founder and CEO of AVE strategic consulting.