Bile acids help facilitate intestinal lipid absorption and have endocrine activity in glucose, lipid and bone metabolism. Obesity and exercise influence bile acid metabolism and have opposite effects in bone. This study investigates if regular exercise helps mitigate the adverse effects of obesity on bone, potentially by reversing alterations in bile acid metabolism. Four-month-old female Sprague Dawley rats either received a high-fat diet (HFD) or a chow-based standard diet (lean controls). During the 10-month study period, half of the animals performed 30 min of running at moderate speed on five consecutive days followed by two days of rest. The other half was kept inactive (inactive controls). At the study’s end, bone quality was assessed by microcomputed tomography and biomechanical testing. Bile acids were measured in serum and stool. HFD feeding was related to reduced trabecular (−33%, p = 1.14 × 10−7) and cortical (−21%, p = 2.9 × 10−8) bone mass and lowered femoral stiffness (12–41%, p = 0.005). Furthermore, the HFD decreased total bile acids in serum (−37%, p = 1.0 × 10−6) but increased bile acids in stool (+2-fold, p = 7.3 × 10−9). These quantitative effects were accompanied by changes in the relative abundance of individual bile acids. The concentration of serum bile acids correlated positively with all cortical bone parameters (r = 0.593–0.708), whilst stool levels showed inverse correlations at the cortical (r = −0.651–−0.805) and trabecular level (r = −0.656–−0.750). Exercise improved some trabecular and cortical bone quality parameters (+11–31%, p = 0.043 to 0.001) in lean controls but failed to revert the bone loss related to the HFD. Similarly, changes in bile acid metabolism were not mitigated by exercise. Prolonged HFD consumption induced quantitative and qualitative alterations in bile acid metabolism, accompanied by bone loss. Tight correlations between bile acids and structural indices of bone quality support further functional analyses on the potential role of bile acids in bone metabolism. Regular moderate exercise improved trabecular and cortical bone quality in lean controls but failed in mitigating the effects related to the HFD in bone and bile acid metabolism.

Background Previously, we could show that zonulin, a marker for intestinal permeability, is linked to a higher risk of mortality in cirrhotic patients. Microbiome analysis found that Phascolarctobacterium is associated with improved intestinal permeability and lower mortality. We aim to verify these results in a second, independent study cohort.

Objectives: Calibration is an important source of variability in liquid chromatography mass spectrometry (LC-MS) methods for insulin-like growth factor 1 (IGF-1). This study investigated the impact of different calibrator matrices on IGF-1 measurements by LC-MS. Moreover, the comparability of immunoassays and LC-MS was assessed.Design & Methods: Calibrators from 12.5 to 2009 ng/ml were prepared by spiking WHO international Standard (ID 02/254 NIBSC, UK) into the following matrices: native human plasma, fresh charcoal-treated human plasma (FCTHP), old charcoal-treated human plasma, deionized water, bovine serum albumin (BSA), and rat plasma (RP). A validated in-house LC-MS method was calibrated repeatedly with these calibrators. Then, serum samples from 197 growth hormone excess and deficiency patients were analysed with each calibration.Results: The seven calibration curves had different slopes leading to markedly different patient results. The largest differences in IGF-1 concentration from the median (interquartile range) was observed with the calibrator in water and the calibrator in RP (336.4 [279.6-417.0] vs. 112.5 [71.2-171.2], p < 0.001). The smallest difference was observed with calibrators in FCTHP and BSA (141.8 [102.0-198.5] vs. 127.9 [86.9-186.0], p < 0.049). Compared to LC-MS with calibrators in FCTHP, immunoassays showed relevant proportional bias (range:-43% to-68%), constant bias (range: 22.84 to 57.29 ng/ml) and pronounced scatter. Comparing the immunoassays with each other revealed proportional bias of up to 24%.Conclusions: The calibrator matrix is critical for the measurement of IGF-1 by LC-MS. Regardless of the calibrator matrix, LC-MS shows poor agreement with immunoassays. Also, the agreement between different immunoassays is variable.

Abstract Background Sarcopenia in liver cirrhosis is associated with low quality of life and high mortality risk. The pathogenesis has yet to be fully understood. We hypothesized that gut microbiome, bile acid (BA) composition and metabolites differ between cirrhotic patients with and without sarcopenia and contribute to pathogenesis. Methods Cirrhotic patients with (n = 78) and without (n = 38) sarcopenia and non‐cirrhotic controls with (n = 39) and without (n = 20) sarcopenia were included in this study. Faecal microbiome composition was studied by 16S rDNA sequencing, serum and faecal BA composition by ultra‐high‐performance liquid chromatography–tandem mass spectrometry, and metabolite composition in serum, faeces and urine by nuclear magnetic resonance. Results Bacteroides fragilis, Blautia marseille, Sutterella spp. and Veillonella parvula were associated with cirrhotic patients with sarcopenia, whereas Bacteroides ovatus was more abundant in cirrhotic patients without sarcopenia. We observed significantly elevated secondary BAs, deoxycholic acid (DCA; P = 0.01) and lithocholic acid (LCA; P = 0.02), and the ratios of deoxycholic acid to cholic acid (DCA:CA; P = 0.04), lithocholic acid to chenodeoxycholic acid (LCA:CDCA; P = 0.03) and 12 alpha‐hydroxylated to non‐12 alpha‐hydroxylated BAs (12‐α‐OH:non‐12‐α‐OH BAs; P = 0.04) in serum of cirrhotic patients with sarcopenia compared with cirrhotic patients without sarcopenia, indicating an enhanced transformation of primary to secondary BAs by the gut microbiome. CA (P = 0.02) and the ratios of CA:CDCA (P = 0.03) and total ursodeoxycholic acid to total secondary BAs (T‐UDCA:total‐sec‐BAs, P = 0.03) were significantly reduced in the stool of cirrhotic patients with sarcopenia compared with cirrhotic patients without sarcopenia. Also, valine and acetate were significantly reduced in the serum of cirrhotic patients with sarcopenia compared with cirrhotic patients without sarcopenia (P = 0.01 and P = 0.03, respectively). Multivariate logistic regression further confirmed the association of B. ovatus (P = 0.01, odds ratio [OR]: 12.8, 95% confidence interval [CI]: 168.1; 2.2), the ratios of 12‐α‐OH:non‐12‐α‐OH BAs (P = 0.03, OR: 2.54, 95% CI: 0.99; 6.55) and T‐UDCA:total‐sec‐BAs (P = 0.04, OR: 0.25, 95% CI: 0.06; 0.98) in serum and stool CA:CDCA (P = 0.04, OR: 0.79, 95% CI: 0.62; 0.99), and serum valine (P = 0.04, OR: 1.00, 95% CI: 1.02; 1.00) with sarcopenia in cirrhosis after correcting for the severity of liver disease and sex. Conclusions Our study suggests a potential functional gut microbiome–host interaction linking sarcopenia with the altered gut microbiomes, BA profiles and amino acids pointing towards a potential mechanistic interplay in understanding sarcopenia pathogenesis.

Bile acids are a key mediator of the molecular microbiome-host interaction, and various mass spectrometry-based assays have been developed in the recent decade to quantify a wide range of bile acids. We compare existing methodologies to harmonize them. Methodology for absolute quantification of bile acids from six laboratories in Europe were compared for the quantification of the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) and conjugated products glycocholic acid (GCA) and taurocholic acid (TCA). For the bacterially modified secondary bile acids, the quantification of deoxycholic acid (DCA) and lithocholic acid (LCA) was compared. For the murine bile acids, we used the primary muricholic acids (α-MCA and, β-MCA) and the intestinally produced secondary bile acid muricholic (ω-MCA). The standards were spiked into methanol:water (1:1) mix as well as in human and murine serum at either low concentration range (150–3000 nM) or high concentration range (1500–40,000 nM). The precision was better for higher concentrations. Measurements for the hydrophobic unconjugated bile acids LCA and ω-MCA were the most challenging. The quality assessments were generally very similar, and the comprehensive analyses demonstrated that data from chosen locations can be used for comparisons between studies.

Aims Statin treatment did not reduce the risk of cardiovascular events in haemodialysis patients in the 4D and AURORA trials. Post hoc analyses in the 4D study suggested that high cholesterol absorption was associated with increased cardiovascular risk and that atorvastatin would reduce cardiovascular risk in haemodialysis patients with low cholesterol absorption but not in those with high cholesterol absorption. Methods and results AURORA is a randomized, double-blind, placebo-controlled, multi-centre trial in haemodialysis patients. The participants were randomly assigned to receive either rosuvastatin, 10 mg daily, or a matching placebo. There was a follow-up for cardiovascular death with a median duration of 3.9 years. The cholestanol and lathosterol to cholesterol ratios were used to estimate cholesterol absorption and synthesis, respectively. Measurement of non-cholesterol sterols was available in 2332 participants of the 2733 patients included in the primary analysis of the AURORA study. A total of 598 participants died from cardiovascular diseases. The 3rd vs. the 1st tertile of the cholestanol-to-cholesterol ratio was significantly associated with increased risk of cardiovascular death [hazard ratio, HR (95% confidence interval, CI) = 1.36 (1.11-1.65)] in univariate (P = 0.002) and multivariate models (P = 0.034). In contrast, the 3rd vs. the 1st tertile of the lathosterol-to-cholesterol ratio was significantly associated with decreased risk of cardiovascular death [HR (95% CI) = 0.81 (0.67-0.99)] in univariate (P = 0.041) and multivariate (P = 0.019) models. There was no significant interaction between the cholestanol and lathosterol to cholesterol tertiles and treatment group in predicting cardiovascular death. Conclusion The present data from the AURORA study confirm that high cholesterol absorption is associated with increased cardiovascular risk in haemodialysis patients. Assessment of the individual cholesterol absorption rate to guide initiation of statin treatment is not supported by the findings in the AURORA study.

Dear Editor, Neutrophil dysfunction is common in cirrhotic patients1 and is associated with mortality.2 Given the highly increased bile acid (BA) levels in liver cirrhosis3 and their previously shown anti-inflammatory effects,4, 5 we studied if BAs contribute to the development of cirrhosis-associated neutrophil dysfunction. We analysed phagocytosis and reactive oxygen species (ROS) production of neutrophils and serum BAs in cirrhotic patients (n = 109) and healthy controls (n = 21) (Table 1, Tables S3–S4, Supplementary Material and Methods). The cirrhotic cohort had a larger proportion of non-phagocytosing neutrophils in whole blood (Figure S1A,B) and dysregulated ROS production with more neutrophils from cirrhotics producing ROS without a stimulus or in response to N-formyl-met-leu-phe (fMLF), and less neutrophils from cirrhotics producing ROS in response to E. coli compared to healthy controls (Figure S1C–E). The level of intracellular ROS produced by these neutrophils was not significantly different between the groups (Figure S2A–C). Serum BA composition was also different in cirrhotic patients compared to healthy controls (Figures S3A,B and S4A,B). The relative abundance of total deoxycholic acid (DCA) in cirrhotic patients was lower than in healthy controls (Figure S3C, Table S5). Secondary, unconjugated and glycine-conjugated BAs were significantly less abundant in cirrhotic patients’ sera compared to healthy sera. Taurine-conjugated BAs were significantly more abundant in cirrhotic sera compared to healthy sera (Figure S3D–G, Table S5). Neutrophil function and BA composition were significantly different between the groups according to liver cirrhosis severity and aetiology as shown by univariate (Figures S5A–E and S6A–E, Tables S6 and S7) and multivariate analyses (Figures S7A–D, S8A–F and S9A–F). The phagocytic capacity of neutrophils was lowest in hepatitis C patients (Figure S6B), who had the highest relative abundance of total chenodeoxycholic acid (CDCA) in serum (Table S7). Further analysis showed that higher total CDCA relative abundance and lower ursodeoxycholic acid (UDCA) relative abundance could significantly predict the decreased neutrophil phagocytosis in cirrhotic patients. Lower UDCA relative abundance could also predict the dysfunctional ROS production in response to E. coli (Figure S10A–C, Table S8). In order to validate the findings observed with clinical samples in vitro, we treated neutrophils from healthy donors with an artificial ‘BA mix’ that mimicked the BA composition in cirrhotic cohort, as well as with individual or total (sum of unconjugated, taurine and glycine conjugated forms) BAs in pathophysiologically relevant concentrations (Figures 1A, 2A and 3A, Tables S1 and S2, Supplementary Material and Methods). Cytotoxicity of BAs was ruled out (Figure S11). In our experiments ‘BA mix’ significantly induced ROS production by unstimulated neutrophils (Figure 1B,C). ROS production in response to fMLF or E. coli, however, was not changed after treatment with ‘BA mix’ (Figure S14A,B). Further experiments with individual BAs revealed that total LCA, but not other BAs, caused increased ROS production (Figure 1D,E, Figure S12A), with glycolithocholic acid having the most pronounced effect (Figure S13A). LCA has been shown to bind Takeda G protein-coupled Receptor 5, whose activation can trigger ROS production,6, 7 which may explain the effects of LCA and its conjugates. Total CDCA and total LCA reduced ROS production in response to fMLF (Figure 2B,D,F), but not their unconjugated and conjugated forms individually (Figure S13B,C). CDCA has been previously described to inhibit neutrophil chemotaxis and calcium flux, via competing with fMLF for binding to the Formyl Peptide Receptor 1 (FPR1).8 The decreased ROS production caused by pre-incubation with total LCA might be a consequence of cell exhaustion following the LCA-mediated ROS release. Total CDCA, its unconjugated and conjugated forms individually, as well as total DCA caused a decrease in ROS production in response to E. coli (Figure 2C,E,G, Figure S13D,E). DCA has previously been described to inhibit neutrophil chemotaxis and calcium mobilisation and is also thought to be able to inhibit FPR1 signalling.9 There were no significant changes in ROS production after the treatment with total cholic acid (CA) and total UDCA (Figure S12A–C). Reduction in ROS production in response to fMLF and E. coli caused by BAs may contribute to an explanation for the reduced ROS production, which has been shown previously in neutrophils from cirrhotic patients.10 We also tested if treatment with BAs influence phagocytosis in vitro (Figure 3A). ‘BA mix’ did not significantly affect neutrophil phagocytosis (Figure 3B); however, total CDCA and total DCA (but neither their unconjugated and conjugated forms individually, nor total CA, total UDCA and total LCA) significantly reduced phagocytosis of E. coli (Figure 3C,D; Figures S15A,B and S16A–C). The inhibitory effects of total CDCA and total DCA were reversible – neutrophil phagocytosis was not inhibited anymore after the BAs were washed out from the assay medium (Figure 3E–G). In conclusion, we performed an extensive analysis of neutrophil function and serum BA composition in a large cohort of cirrhotic patients. We show that BAs are associated with neutrophil dysfunction in the clinical data from cirrhotic patients, as well as in the experiments where we directly treated neutrophils with BAs. These findings should be validated in patients with more severe stages of cirrhosis, and the molecular mechanisms of the observed BA effects on neutrophil function need to be explored in further studies. The reversibility of neutrophil dysfunction caused by BAs, which we observed, suggests that therapeutical modulation of serum BA profile toward a ‘healthy’ composition could potentially not only prevent neutrophil dysfunction but also restore already impaired neutrophil function in cirrhotic patients. This in turn will allow reducing bacterial infections and associated morbidity and mortality in liver cirrhosis. BA profile modulation may be achieved by oral intake of BAs or modified BAs and by targeting the gut microbiome involved in BA metabolism. The authors thank Bianca Schmerboeck for the technical assistance, and Utsa Karmakar and Julia Chu (CIR, University of Edinburgh) for helpful discussions. They thank Kellie McDonald for English language proofreading of the manuscript and the Computational Bioanalytics core facility (ZMF, Medical University Graz) for help with statistics as well as the Austrian Science Fund (FWF), the Medical University Graz and the Doctoral College ‘Molecular fundamentals of inflammation’ (W1241) for the provision of funding and facilities. All authors have nothing to disclose. Austrian Science Fund FWF (W1241) and the Medical University Graz through the PhD Program Molecular Fundamentals of Inflammation (DK-MOLIN); Medical Research Council (MRC) MR/S008020/1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Background: The cerebral composition of ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) is believed to influence cognitive function and structural damage of the aging brain. However, existing data is inconsistent. Materials and Methods: This retrospective study explored the association between free plasma PUFA concentrations, cognitive function and brain structure atrophy in a well-characterized community-dwelling cohort of elderly individuals without stroke and dementia. Ten different fatty acids were analyzed in stored plasma samples from 391 non-demented elderly individuals by gas chromatography mass spectrometry. Neuropsychiatric tests capturing memory, executive function and visuopractical skills were performed in all participants. Brain atrophy was assessed by MRI in a subset of 167 individuals. Results: Higher plasma concentrations of free ω-6 PUFAs (p = 0.042), and, in particular, linoleic acid (p = 0.01), were significantly associated with lower executive function. No significant association existed between ω-3 PUFA concentrations and cognitive functioning. The volume of the frontal lobes was inversely associated with ω-6 PUFAs, whereas ω-3 PUFAs were positively related with temporal lobe volumes. All associations did not withstand correction for multiple comparisons. Conclusions: Our study suggests subtle effects of PUFA imbalances on cognition and brain structure. Yet the observed associations are weak and unlikely to be of clinical relevance. The brain regions that seem to be most sensitive to imbalances of ω-3 and ω-6 PUFAs are the frontal and temporal lobes.

INTRODUCTION:Bile acid signaling has been suggested to promote BAT activity in various experimental models. However, little is known if and how physiologic bile acid metabolism is linked to BAT function in humans. Here we investigated the association between BAT activity and circulating bile acid concentrations in lean and obese individuals. METHODS:BAT 18F-fluorodeoxyglucose uptake was measured after a standardized cooling protocol by positron emission tomography/computed tomography. Cold-induced thermogenesis was assessed by indirect calorimetry. Fasting bile acid concentrations were determined by high performance liquid chromatography-high-resolution mass spectrometry. RESULTS:In a cohort of 24 BAT-negative and 20 BAT-positive individuals matched by age, sex, and body mass index, circulating bile acid levels were similar between groups except for higher ursodeoxycholic acid and a trend towards a lower 12α-OH/non-12α-OH bile acid ratio in lean participants with active BAT compared to those without. Moreover, the 12α-OH/non-12α-OH ratio, a marker of CYP8B1 activity, correlated negatively with BAT volume and activity. CONCLUSION:Fasting concentrations of major bile acids are not associated with cold-induced BAT activity in humans. However, the inverse association between BAT activity and 12α-OH/non-12α-OH ratio may suggest CYP8B1 as a potential new target in BAT function and warrants additional investigation.

Cholestasis might lead to an impairment of adrenal function as suggested by in vitro and in vivo data as well as by clinical findings. Bile acid and adrenal steroid metabolism not only share the receptors farnesoid X receptor (FXR) and the G protein-coupled bile acid receptor 1 (TGR5), but supraphysiological bile acid levels were found to stimulate steroidogenesis independent of FXR and TGR5. Our previous experimental findings revealed that mice fed bile acids or subjected to common bile duct ligation develop hypercortisolemia. We thus aimed to assess adrenal gland function in patients with cholestasis. Adrenal gland function was assessed in 36 patients with cholestasis and in 32 patients without cholestasis by measuring total serum cortisol, adrenocorticotropic hormone (ACTH), as well as the increase of cortisol 20 and 30 min after administration of 1 µg of ACTH. Bile acid levels and bile acid pool composition were determined by high-resolution mass spectrometry. Patients with cholestasis per definition had markedly elevated levels of alkaline phosphatase (AP), bilirubin and serum bile acids. Baseline cortisol and maximum cortisol after ACTH stimulation were significantly higher in patients with cholestasis compared to controls. Increase of cortisol after ACTH stimulation and ACTH did not differ. In the cholestasis group, baseline cortisol correlated with bilirubin but not with AP, total serum bile acids and levels of conjugated and unconjugated bile acid species. Patients with duration of cholestasis < 6 months (n = 30) had significantly higher baseline cortisol levels than those with long standing cholestasis (> 6 months), together with higher bilirubin levels. We find no evidence of adrenal insufficiency in non-cirrhotic patients with cholestasis. In contrast, patients with cholestasis show hypercortisolism associated with disease severity as mirrored by levels of bilirubin. Lack of ACTH increase in cholestasis suggests a direct effect of cholestasis on adrenals and not on the pituitary gland. Further studies are needed to elucidate the mechanism of cortisol elevation in patients with cholestasis and its clinical significance.

IsoBAs, stereoisomers of primary and secondary BAs, are found in feces and plasma of human individuals. BA signaling via the nuclear receptor FXR is crucial for regulation of hepatic and intestinal physiology/pathophysiology. Aim: Investigate the ability of BA-stereoisomers to bind and modulate FXR under physiological/pathological conditions. Methods: Expression-profiling, luciferase-assays, fluorescence-based coactivator-association assays, administration of (iso)-BAs to WT and cholestatic mice. Results: Compared to CDCA/isoCDCA, administration of DCA/isoDCA, UDCA/isoUDCA only slightly increased mRNA expression of FXR target genes; the induction was more evident looking at pre-mRNAs. Notably, almost 50% of isoBAs were metabolized to 3-oxo-BAs within 4 h in cell-based assays, making it difficult to study their actions. FRET-based real-time monitoring of FXR activity revealed that isoCDCA>CDCA stimulated FXR, and isoDCA and isoUDCA allowed fully activated FXR to be re-stimulated by a second dose of GW4064. In vivo co-administration of a single dose of isoBAs followed by GW4064 cooperatively activated FXR, as did feeding of UDCA in a background of endogenous FXR ligands. However, in animals with biliary obstruction and concomitant loss of intestinal BAs, UDCA was unable to increase intestinal Fgf15. In contrast, mice with an impaired enterohepatic circulation of BAs (Asbt−/−, Ostα−/−), administration of UDCA was still able to induce ileal Fgf15 and repress hepatic BA-synthesis, arguing that UDCA is only effective in the presence of endogenous FXR ligands. Conclusion: Secondary (iso)BAs cooperatively activate FXR in the presence of endogenous BAs, which is important to consider in diseases linked to disturbances in BA enterohepatic cycling.

Correction to:Gut microbiome composition in liver cirrhosis is associated with serum bile acid profileZ Gastroenterol 2021; 59(08): P88DOI: 10.1055/s-0041-1734326

Summary Background The PX-104 is an oral non-steroidal agonist for the farnesoid X receptor (FXR), a key regulator of bile acid (BA), glucose and lipid homeostasis. Aims and methods This single center, proof of concept study evaluated the efficacy, safety and tolerability of PX-104 in non-diabetic NAFLD patients. 12 individuals were treated daily with 5 mg of PX-104 orally for 4 weeks. Serum liver enzymes, insulin sensitivity by clamp like index (CLIX) and hepatic fat by proton 1H‑MRS, MRI-PDFF and CAP were assessed. Hepatic energy metabolism and Kupffer cell function were evaluated by phosphorus 31P‑MRS and superparamagnetic iron oxide MRI (SPIO-MRI). Other readouts included serum lipids and markers of BA metabolism/signaling besides fecal microbiome and BA analysis. Results A significant decrease in ALT (p = 0.027; 1‑tailed) and GGT (p = 0.019) was observed, without changes in serum alkaline phosphatase or serum lipids. Insulin sensitivity improved in 92% of patients (p = 0.02). However, hepatic steatosis measured by PDFF-MRI, 1H‑MRS and CAP besides extended serum lipoprotein and BA profiles did not change. NADPH/γATP ratios at 31P‑MRS significantly decreased (p = 0.022) possibly reflecting reduced hepatic inflammatory stress, but SPIO-MRI remained unchanged. Reduced preponderance of Coriobacteriaceae (p = 0.036) correlated with a relative reduction of total fecal BAs. There were no serious adverse events but short intervals of cardiac arrhythmia recorded in 2 patients led to termination of the study. Conclusion The non-steroidal FXR agonist PX-104 improved insulin sensitivity and liver enzymes after 4 weeks of treatment in non-diabetic NAFLD patients. Changes in fecal BAs and gut microbiota deserve more extensive investigations.

Secondary sclerosing cholangitis in critically ill patients (SC-CIP) occurs after long-term intensive care treatment. This study aimed to assess the gut-liver axis in SC-CIP. Stool microbiome composition, gut permeability, bacterial translocation and serum bile acid profiles of 18 SC-CIP patients compared to 11 patients after critical illness without liver disease (CIP controls), 21 patients with cirrhosis and 21 healthy controls were studied. 16S rDNA was isolated from stool and sequenced using the Illumina technique. Diamine oxidase, zonulin, soluble CD14 (sCD14) and lipopolysaccharide binding protein were measured in serum and calprotectin in stool. Serum bile acids were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS). Reduced microbiome alpha diversity and altered beta diversity were seen in SC-CIP, CIP controls and cirrhosis compared to healthy controls. SC-CIP patients showed a shift towards pathogenic taxa and an oralization. SC-CIP, CIP controls and cirrhotic patients presented with impaired gut permeability, and biomarkers of bacterial translocation were increased in SC-CIP and cirrhosis. Total serum bile acids were elevated in SC-CIP and cirrhosis and the bile acid profile was altered in SC-CIP, CIP controls and cirrhosis. In conclusions, observed alterations of the gut-liver axis in SC-CIP cannot solely be attributed to liver disease, but may also be secondary to long-term intensive care treatment.

Antiquitin (ATQ) deficiency leads to tissue, plasma, and urinary accumulation of alpha-aminoadipic semialdehyde (AASA) and its Schiff base delta-1-piperideine-6-carboxylate (P6C). Although genetic testing of ALDH7A1 is the most definitive diagnostic method, quantifications of pathognomonic metabolites are important for the diagnosis and evaluation of therapeutic and dietary interventions. Current metabolite quantification methods use laborious, technically highly complex, and expensive liquid chromatography-tandem mass spectro-metry, which is available only in selected laboratories worldwide. Incubation of ortho-aminobenzaldehyde (oABA) with P6C leads to the formation of a triple aromatic ring structure with characteristic absorption and fluorescence properties. The mean concentration of P6C in nine urine samples from seven ATQ-deficient patients under standard treatment protocols was statistically highly significantly different (P < .001) compared to the mean of 74 healthy controls aged between 2 months and 57 years. Using this limited data set the specificity and sensitivity is 100% for all tested age groups using a P6C cut-off of 2.11 μmol/mmol creatinine, which represents the 99% prediction interval of the P6C concentrations in 17 control urine samples from children below 6 years of age. Plasma P6C concentrations were only elevated in one ATQ subject, possibly because P6C is trapped by pyridoxal-5-phosphate (PLP) blocking fusing with oABA. Nevertheless, both urine and plasma samples were amenable to the quantification of exogenous P6C with high response rates. The P6C quantification method using fusion of oABA with P6C is fast, simple, and inexpensive and might be readily implemented into routine clinical diagnostic laboratories for the early diagnosis of neonatal pyridoxine-dependent epilepsy.