carbocyanines and Liver-Diseases

Reliable ammonia quantification assays are essential for monitoring ammonemia in patients with liver diseases. In this study, we describe the development process of a microplate-based assay for accurate, precise, and robust ammonia quantification in biological fluids, following regulatory guidelines on bioanalytical method validation. The assay is based on transmembrane pH-gradient polymersomes that encapsulate a pH-sensitive ratiometric fluorophore, the fluorescence signal of which correlates with the ammonia concentration in the sample. Using a four-parameter logistic regression, the assay had a large quantification range (30-800 μM ammonia). As for selectivity, the presence of amino acids or pyruvate (up to clinically relevant concentrations) showed no assay interference. In samples with low bilirubin levels, polymersomes containing the fluorophore pyranine provided accurate ammonia quantification. In samples with high bilirubin concentrations, billirubin's optical interference was alleviated when replacing pyranine with a close to near-infrared hemicyanine fluorophore. Finally, the assay could correctly retrieve the ammonia concentration in ammonia-spiked human plasma samples, which was confirmed by comparing our measurements with the data obtained using a commercially available point-of-care device for ammonia.

Topics: Ammonia; Carbocyanines; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Liver Diseases; Spectrometry, Fluorescence

Sepsis causes mitochondrial oxidative injury and swelling. Ethyl pyruvate (EP) is a cytoprotective agent, while aquaporin-8 (AQP8) is a mitochondrial water channel that can induce mitochondrial swelling. We assessed whether EP protects mitochondria during sepsis, and whether AQP8 contributes to the underlying mechanisms. A cecal ligation and puncture (CLP) sepsis model was established in Sprague-Dawley rats, randomized to 3 groups: sham (n=20), CLP (n=59) and CLP+EP (n=51). All rats received postoperative intraperitoneal fluid resuscitation (30 ml/kg); the CLP+EP group also received intraperitoneal EP (100 mg/kg). Survival was assessed at 24 hours. Hepatic mitochondrial ultrastructure was characterized by electron microscopy. The membrane potential of isolated hepatic mitochondria was determined using JC-1 and flow cytometry. Mitochondrial AQP8 expression and cytochrome C (Cyt C) release were measured by Western blotting (values normalized to ß-actin). Survival in the sham, CLP and CLP+EP groups was 100%, 21% and 41%, respectively. Mitochondrial cross-sectional area was smaller in the CLP+EP group than in the CLP group (0.231±0.110 vs. 0.641±0.460 µm(2); P<0.001), with a tendency for a lower form factor (a measure of contour irregularity) in the CLP+EP group. Mitochondrial depolarization by CLP was inhibited by EP. Mitochondrial Cyt C release was higher in the CLP group than in the sham (1.211±0.24 vs. 0.48±0.03) or CLP+EP (0.35±0.39) groups. AQP8 expression was similar between groups, with a trend for lower expression in the CLP+EP group compared with the CLP group. EP improves sepsis outcome by targeting the mitochondrion, possibly through modulation of AQP8 expression.

Topics: Animals; Aquaporins; Benzimidazoles; Carbocyanines; Disease Models, Animal; Humans; Liver; Liver Diseases; Male; Mitochondria, Liver; Mitochondrial Swelling; Pyruvates; Random Allocation; Rats; Rats, Sprague-Dawley; Sepsis