Plasma bilirubin and (un)conjugated bile acid levels in patients on the day of diagnosis of severe sepsis.

<p>(A) The plot depicts median log₂ fold changes of bilirubin (Bili), and unconjugated as well as glycine- and taurine-conjugated bile acid quantities in plasma of severely septic patients (<i>n</i> = 48) fulfilling American College of Chest Physicians/Society of Critical Care Medicine consensus criteria compared to non-septic controls (<i>n</i> = 20) (*<i>p</i><0.05 compared to controls). (B) Receiver operating characteristics of bilirubin, CDCA, TDCA, or the combined performance of CDCA+TDCA in predicting 28-d mortality. DCA, deoxycholic acid; GDCA, glycodeoxycholic acid; GCDCA, glycochenodeoxycholic acid; GLCA, glycolithocholic acid; GLCAS, glycolithocholic acid sulphate; GUDCA, glycoursodeoxycholic acid; TCDCA, taurochenodeoxycholic acid; TLCA, taurolithocholic acid; TLCAS, taurolithocholic acid sulphate; TUDCA, tauroursodeoxycholic acid; UDCA, ursodeoxycholic acid.</p

Clinical characteristics of septic patients subjected to targeted metabolomic analysis of bile acids.

<p>APACHE II, Acute Physiology and Chronic Health Evaluation II; SAPS II, Simplified Acute Physiology Score; SOFA, Sequential Organ Failure Assessment.</p

Polymicrobial sepsis severely impairs the activity of enzymes responsible for phase I and II biotransformation.

<p>(A) Activities of CYP1A, CYP2A, CYP2B, CYP2C, and CYP2E were assessed by ethoxycoumarin O-deethylation, while (B) CYP3A activity was quantified using the ethylmorphine N-demethylation model reaction for phase I biotransformation. Glutathione-S-transferase activity (C) and bilirubin glucuronidation (D), representing typical phase II conjugation reactions, were assessed by the model reaction 1-chloro-2,4-dinitrobenzene conjugation, resulting in the formation of dinitrobenzene-glutathione conjugate (GS-DNB), or the Burchell method, respectively (<i>n</i> = 5 for sham, <i>n</i> = 8 for sepsis, *<i>p</i> = 0.004 compared to sham).</p

Polymicrobial sepsis causes deranged bile acid conjugation and transport.

<p>At 15 h after sepsis induction, plasma, liver tissue, and bile were subjected to targeted metabolomics. Expression of BAAT, facilitating conjugation to taurine and glycine, was quantified by immunoblotting. (A) The plot depicts median log₂ fold changes of unconjugated as well as glycine- and taurine-conjugated bile acid in plasma, liver, and bile, comparing septic to sham-operated rats (<i>n</i> = 12 per group, *<i>p</i><0.05 or **<i>p</i><0.01 compared to sham). (B) Conjugation index as a surrogate for the observed conjugation defect reflected by the ratio of unconjugated bile acids CA and CDCA to the corresponding taurine (TCA and taurochenodeoxycholic acid) and glycine (GCA and glycochenodeoxycholic acid) conjugates in plasma, liver and bile (ratio given as log₂ fold change, <i>n</i> = 12 per group). (C and D) Representative immunoblots of BAAT 15 h after sepsis induction in cytosolic (c) as well as peroxisomal (p) fractions, with corresponding densitometric analysis (<i>n</i> = 5 for sham, <i>n</i> = 8 for sepsis; BAAT (c): *<i>p</i> = 0.002; BAAT (p): *<i>p</i> = 0.006 compared to sham). Densitomentric values are normalised to β-actin. DCA, deoxycholic acid; GDCA, glycodeoxycholic acid; GCDCA, glycochenodeoxycholic acid; GLCA, glycolithocholic acid; GLCAS, glycolithocholic acid sulphate; GUDCA, glycoursodeoxycholic acid; TCDCA, taurochenodeoxycholic acid; TLCA, taurolithocholic acid; TLCAS, taurolithocholic acid sulphate; TUDCA, tauroursodeoxycholic acid; UDCA, ursodeoxycholic acid.</p

Assessment of markers for hepatocellular injury and sepsis-associated cholestasis at 15 h after infection.

<p>Markers were assessed in serum or bile, as specified.</p>*<p><i>p</i><0.05 or</p>**<p><i>p</i><0.01 compared to sham; <i>n</i> = 12 animals for each condition.</p><p>n.d., not detectable.</p

Rat model allowing assessment of early changes in gene expression, and relationships to outcome.

<p>(A) Protocol for instrumentation, induction of sepsis, echo-guided prognosis stratification, and tissue harvesting. (B) The heatmap displays expression profiles of 1,373 genes with significantly different expression levels among the naïve, sham, predicted sepsis survivor, and predicted non-survivor groups (<i>n</i> = 4 per group). <i>k</i>-Means clustering revealed four gene clusters with comparable behaviour. Associated colours represent variance-normalised expression for individual genes: red colour intensity indicates a relative expression greater than the mean, blue indicates the opposite. (C) The transcripts identified in (B) were analysed by Ingenuity Pathway Analysis to identify canonical pathways found to be up- or down-regulated in animals predicted to die. Presented is a bar chart of the most statistically significant (corresponding to <i>p</i>-value) down-regulated (blue) and up-regulated (red) pathways associated with poor prognosis. Numbers in parentheses represent the number of differentially regulated transcripts involved in a defined pathway.</p

Altered expression of canalicular bile acid and organic anion transporters in sepsis.

<p>(A) Distribution and localisation pattern of Bsep and Mrp2 in livers obtained from control and septic rats using confocal microscopy. Na,K-ATPase was used to stain the basolateral plasma membrane. Staining for Bsep revealed a substantial decrease in protein expression after sepsis induction. In sham-treated animals immunoreactive Mrp2 delineates bile canaliculi. The Mrp2 staining pattern in septic rats is irregular, disrupted, and accompanied by recovery of Mrp2 inside the cell, as shown by punctuate staining in the pericanalicular region. Lowermost panels represent merged pictures of Na,K-ATPase and Mrp2. Scale bar: 20 µm. (B) Electron micrographs of freeze-fracture-immunolabelled Mrp2 protein in the plasma membrane of isolated hepatocytes at 15 h post-infection to further study irregular staining observed in sepsis. The cell surface of sham-treated animals is densely covered with microvilli, visible as cross-fractured stubs (stars). A lengthwise fracture of the microvilli membrane exposes a dense labelling with Mrp2 protein arranged in small clusters along microvilli (white arrowheads). By contrast, hepatocytes isolated from septic animals exhibited a loss of microvilli, while Mrp2 was predominantly found in vesicles located closely beneath the canalicular membrane (black arrowheads). The inset shows a top view of a bile canaliculus formed by two adjacent hepatocytes (scale bar: 1 µm). Scale bars in upper and lower panels indicate 200 nm and 100 nm, respectively. EF, exoplasmic fracture face; PF, protoplasmic fracture face.</p

Sepsis-induced excretory dysfunction, as visualised by accumulation of the xenobiotic indocyanine green and bilirubin.

<p>(A) Non-invasive whole body near-infrared fluorescence imaging of ICG at 15 h after sepsis induction. Shown are representative images from sham-treated animals (left panels) and animals subjected to lethal sepsis (right panels) for five biological replicates each. While ICG is eliminated via hepatobiliary excretion into the duodenum within the 300-min observation period in sham animals, the dye accumulates in the livers of septic animals, with an almost absence of fluorescence signal in the gut, as confirmed after laparotomy (lower panels). The dotted line divides upper and lower abdominal quadrants for orientation. (B) Subsequent epifluorescence microscopic examination of liver surfaces at 300 min after ICG administration in sham and septic animals. Stars indicate central veins (magnification 50×, pseudo-coloured). (C) ICG fluorescence intensities around central veins were significantly higher in septic compared to sham-operated animals (<i>n</i> = 4 animals/group, five perivenous and five periportal areas per animal, *<i>p</i> = 0.031 compared to sham). (D) Representative micro-Raman images of tissue sections from liver sections obtained from sham-operated and septic rats 15 h post-insult. In the livers of the septic rats, relative intensities of the bilirubin component are elevated in the perivenous region (stars). In contrast, only minor local spots of accumulated bilirubin could be verified in livers of control animals. Scale bars: 50 µm. (E) Raman spectrum of the bilirubin component (red trace) with crystalline bilirubin for comparison (black trace). Main spectral contributions of bilirubin are resolved near 971, 1,229, and 1,606 cm⁻¹ (arrows at 971 and 1,229 cm⁻¹; high peak at 1,606 cm⁻¹).</p