Release of total ketones in the supernatants.

<p>Fat loaded primary human hepatocytes that had the defatting treatment showed an increase in cell culture supernatant levels of total ketone bodies of 1.22-fold over 24 hours and 1.40-fold over 48 hours. Data reports the median of three separate experiments performed in quadruplicate and errors bars the interquartile range. Comparisons performed using two-tailed t-test. * = p<0.05.</p

Assessment of the cytotoxicity of the defatting cocktail to human cells of the liver via MTT assay.

<p>Panel A: the toxicity of the defatting cocktail was tested in primary human hepatocytes and results showed a significant improvement of 11% in viability of the defatting treatment group compared with the fatty vehicle control group. Panel B: Treatment of human intra-hepatic endothelial cells (HIEC) with the drugs had no effect on cell viability compared with the control groups. Panel C: treatment of cholangiocytes with the defatting cocktail did not demonstrate any cytotoxic effect to the cells and indicated a slight improvement in viability compared to the control groups. Panels D and E: Phase contrast microscopy showing representative images of HIEC (Panel D) and cholangiocytes (Panel E) at different time points. No gross modifications in cell integrity were observed in either cell type which was consistent and supportive of the MTT data. Data report the median of three separate experiments performed in quadruplicate and errors bars the interquartile range. Comparisons performed using two-tailed t-test. * = p<0.05.</p

Study design.

<p>Series 1: Isolated primary human hepatocytes (PHH) were left in standard media for 2 days and then received media supplemented with fatty acids. After 2 days of fat loading the fatty PHH were allocated to the defatting treatment group where the media was supplemented with the defatting cocktail of drugs, and the control groups, the standard control group and the vehicle control group that received vehicle only. Lean hepatocytes were kept in standard culture conditions throughout the experimental period. The experimentation period lasted for two days thereafter. Series 2: Human intra-hepatic endothelial cells (HIEC) and cholangiocytes were immuno-magnetically separated with Dynabeads conjugated with cell-specific monoclonal antibody. The cells were left in culture for 2 days in standard media to reach confluence and then were allocated to the intervention group that received the defatting cocktail and the control groups, the standard control group and the vehicle control group that had the media supplemented with the vehicle only. The experimentation period lasts for two days thereafter.</p

Defatting of fat loaded primary human hepatocytes (PHH).

<p>Panel A: The positive are of oil red O of the defatting treatment group was reduced by 28% in comparison with the vehicle control group over 24 hours and 54% over 48 hours. Panel B: Intracellular triglyceride levels of the defatting treatment group were reduced by 32% within 24 hours of treatment and 35% within 48 hours, in comparison with the fatty vehicle control group. Panel C: Oil red O staining picture of PHH of the defatting group at the end of the 48 hours of treatment. There is a predominance of small lipid droplets in the cytoplasm of the cells and the nucleus is in its usual position. Series 2: shows a series of oil red O staining pictures from PHH in culture at different time points of the experiments. Panel D shows lean cells in culture, after the incubation with fatty acids they become loaded with fat (panel E). Those fat loaded PHH were then incubated with only the vehicle of the drugs for 48 hours and the lipid content decreased over time (panel F) or had the defatting treatment that showed the significant higher decrease in the area of oil red O (panel G). Data report the median of three separate experiments performed in quadruplicate and errors bars the interquartile range. Comparisons performed using two-tailed t-test. * = p<0.05.</p

Results of fat loading of primary human hepatocytes.

<p>Panel A: The supplementation of the media with the combination of fatty acids resulted in a cell viability rate of 81% after 48 hours of incubation. Panel B: Oil red O staining image of PHH at the end of the fat loading period. There is predominance of large lipid droplets displacing the nucleus of the cells to the periphery (black arrow). Panel C: At the end of 48 hours of fatting load there was a significant increase of 14-fold of the positive area of oil red O. Panel D: Intracellular triglycerides increased 8-fold within 48 hours of incubation with fatty acids. Data report the median of three separate experiments performed in quadruplicate and errors bars the interquartile range. Comparisons performed using two-tailed t-test. * = p<0.05.</p

Principal component analysis scores plots highlighting the metabolic separation of the DBD and DCD grafts in the cold phase (T₁) and separately the post-reperfusion phase (T₂), based on analyses of just the 50 and 64 peaks identified as being significantly different (between DBD and DCD) for the T₁ and T₂ groups, respectively.

<p>Variance explained for T₁, PC1 = 36.75% and PC2 = 19.39% and for T₂, PC1 = 25.22% and PC2 = 17.41%.</p

The differences between tryptophan and kynurenine in failed allografts due to Primary non-function/PNF (n = 2) vs. non-PNF (n = 36) in the cold phase and post reperfusion.

<p>The data show the relative abundances of the metabolites with 95% confidence intervals (statistics not applied due to limited sample size).</p

Donor and recipient demographics and surgical data.

<p>Donor and recipient demographics and surgical data.</p

Metabolic pathways discovered to differ significantly between DCD and DBD grafts, including the associated putatively annotated metabolites in those pathways.

<p>Metabolic pathways discovered to differ significantly between DCD and DBD grafts, including the associated putatively annotated metabolites in those pathways.</p

Top putatively annotated metabolic fold-changes between the DCD and DBD grafts: combined results for the comparison, (i) in the cold phase (T₁), (ii) following reperfusion (T₂), and (iii) in response from going from T₁ to T₂.

<p>The average absolute ppm error was 0.2758, range: 0.0069–0.6835.</p