Abstract
Hyperammonemia is a metabolic condition characterized by elevated levels of ammonia and a common event in acute liver injury/failure and chronic liver disease. Even though hepatic ammonia levels are potential predictive factors of patient outcome, easy and inexpensive methods aiming at the detection of liver ammonia accumulation in the clinical setting remain unavailable. Thus, herein we have developed a morphological method, based on the utilization of Nessler´s reagent, to accurately and precisely detect the accumulation of ammonia in biological tissue. We have validated our method against a commercially available kit in mouse tissue samples and, by using this modified method, we have confirmed the hepatic accumulation of ammonia in clinical and animal models of acute and chronic advanced liver injury as well as in the progression of fatty liver disease. Overall, we propose a morphological method for ammonia detection in liver that correlates well with the degree of liver disease severity and therefore can be potentially used to predict patient outcome.
Highlights
Ammonia is one of the main products of nitrogen metabolism that is usually transported from muscle and other peripheral tissues to the liver to be converted to urea by the urea cycle and excreted by the kidneys in the form of urine
To be able to compare ammonia quantifications between different laboratories and hospital facilities we propose a scoring system, Ammonia Score (AS), as follows: at least 3 samples from healthy livers either human or mouse are used as controls and used for normalization of the rest of the samples; samples are normalized to the average values of these samples and their score classified according to the following criteria (Area ratio between 0-2- Score 1, area ratio between 2-10- Score 2, area ratio between 10-20- Score 3, area ratio between 20-30- Score 4, area ratio between 30-40- Score 5, area ratio >40- Score 6)
Coefficient of variations (C.V.) of the 5 measurements of the images of ammonia staining obtained from each sample are within 1–5% whereas the C.V. between ammonia measurements of 3 different sections obtained from the same healthy mouse liver sample is between 13–41% (n = 3), indicative of good assay precision
Summary
Ammonia is one of the main products of nitrogen metabolism that is usually transported from muscle and other peripheral tissues to the liver to be converted to urea by the urea cycle and excreted by the kidneys in the form of urine. Innovacion 2013-2016 cofinanciado con Fondos FEDER to M.L.M.-C and J.M.M. respectively, Instituto de Salud Carlos III:PIE14/00031, integrado en el Plan Estatal de Investigacion Cientifica y Tecnica y Innovacion 2013-2016 cofinanciado con Fondos FEDER (to M.L.M.-C and J.M.M), EITB. BIO15/CA/014, Asociacion Española contra el Cancer (T.C.D, P.F-T and M.L.M-C). Ciberehd_ISCIII_MINECO is funded by the Instituto de Salud Carlos III. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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