Metabolic Profiling Identifies 1-MetHis and 3-IPA as Potential Diagnostic Biomarkers for Patients With Acute and Chronic Heart Failure With Reduced Ejection Fraction.

BACKGROUND: Metabolomics has become a valuable tool for identifying potential new biomarkers and metabolic profiles. It has the potential to improve the diagnosis and prognosis of different phenotypes of heart failure. To generate a distinctive metabolic profile, we assessed and compared the metabolic phenotypes of patients with acute decompensated heart failure (ADHF), patients with chronic heart failure (CHF), and healthy controls. METHODS: Plasma metabolites were analyzed by liquid-chromatography mass spectrometry/mass spectrometry and the MxP Quant 500 kit in 15 patients with ADHF, 50 patients with CHF (25 with dilated cardiomyopathy, 25 with ischemic cardiomyopathy), and 13 controls. RESULTS: Of all metabolites identified to be significantly altered, 3-indolepropionic acid and 1-methyl histidine showed the highest concentration differences in ADHF and CHF compared with control. Area under the curve-receiver operating characteristic analysis showed an area under the curve ≥0.8 for 3-indolepropionic acid and 1-methyl histidine, displaying good discrimination capabilities between control and patient cohorts. Additionally, symmetrical dimethylarginine (mean, 1.97±0.61 [SD]; P=0.01) was identified as a suitable biomarker candidate for ADHF and kynurenine (mean, 1.69±0.39 [SD]; P=0.009) for CHF when compared with control, both demonstrating an area under the curve ≥0.85. CONCLUSIONS: Our study provides novel insights into the metabolic differences between ADHF and CHF and healthy controls. We here identify new metabolites for potential diagnostic and prognostic purposes.

Contribution of radixin and ezrin to the maintenance of hepatocytes' excretory function in health and disease.

Background & aims: Excretory liver failure is frequently associated with poor prognosis in critically ill patients. It is characterized by the loss of canalicular membrane export pumps at the hepatocyte membrane. The membrane export pump Multidrug resistant-associated protein (MRP) 2 is pivotal in hepatocytes for brushed membrane morphology and transport of various metabolites. In addition, MRP2 anchoring proteins of the Ezrin/Radixin/Moesin (ERM) family are crucial for the correct MRP2 location, integration, and function in different tissues. In hepatocytes, altered ERM signaling is elementary for developing excretory liver failure. Methods: Polarized human HepaRG cells, primary human hepatocytes, and hepatocyte-specific Ezrin knockout mice are employed to investigate ERM expression and function in health and the bile duct ligation model of obstructive cholestasis. Results: ERM-scaffolding protein Ezrin has no relevant function in maintaining the canalicular structure in hepatocytes during health and disease. Conclusions: Homeostasis of the canalicular pole in hepatocytes is maintained exclusively by Radixin but not Ezrin, and Radixin dysfunction promotes cholestasis.

Quantification of Bile Acids in Cerebrospinal Fluid: Results of an Observational Trial.

(1) Background: Bile acids, known as aids in intestinal fat digestion and as messenger molecules in serum, can be detected in cerebrospinal fluid (CSF), although the blood-brain barrier is generally an insurmountable obstacle for bile acids. The exact mechanisms of the occurrence, as well as possible functions of bile acids in the central nervous system, are not precisely understood. (2) Methods: We conducted a single-center observational trial. The concentrations of 15 individual bile acids were determined using an in-house LC-MS/MS method in 54 patients with various acute and severe disorders of the central nervous system. We analyzed CSF from ventricular drainage taken within 24 h after placement, and blood samples were drawn at the same time for the presence and quantifiability of 15 individual bile acids. (3) Results: At a median time of 19.75 h after a cerebral insult, the concentration of bile acids in the CSF was minute and almost negligible. The CSF concentrations of total bile acids (TBAs) were significantly lower compared to the serum concentrations (serum 0.37 µmol/L [0.24, 0.89] vs. 0.14 µmol/L [0.05, 0.43]; p = 0.033). The ratio of serum-to-CSF bile acid levels calculated from the respective total concentrations were 3.10 [0.94, 14.64] for total bile acids, 3.05 for taurocholic acid, 14.30 [1.11, 27.13] for glycocholic acid, 0.0 for chenodeoxycholic acid, 2.19 for taurochenodeoxycholic acid, 1.91 [0.68, 8.64] for glycochenodeoxycholic acid and 0.77 [0.0, 13.79] for deoxycholic acid; other bile acids were not detected in the CSF. The ratio of CSF-to-serum S100 concentration was 0.01 [0.0, 0.02]. Serum total and conjugated (but not unconjugated) bilirubin levels and serum TBA levels were significantly correlated (total bilirubin p = 0.031 [0.023, 0.579]; conjugated bilirubin p = 0.001 [0.193, 0.683]; unconjugated p = 0.387 [-0.181, 0.426]). No correlations were found between bile acid concentrations and age, delirium, intraventricular blood volume, or outcome measured on a modified Rankin scale. (4) Conclusions: The determination of individual bile acids is feasible using the current LC-MS/MS method. The results suggest an intact blood-brain barrier in the patients studied. However, bile acids were detected in the CSF, which could have been achieved by active transport across the blood-brain barrier.

Immunosuppressive effects of circulating bile acids in human endotoxemia and septic shock: patients with liver failure are at risk.

BACKGROUND: Sepsis-induced immunosuppression is a frequent cause of opportunistic infections and death in critically ill patients. A better understanding of the underlying mechanisms is needed to develop targeted therapies. Circulating bile acids with immunosuppressive effects were recently identified in critically ill patients. These bile acids activate the monocyte G-protein coupled receptor TGR5, thereby inducing profound innate immune dysfunction. Whether these mechanisms contribute to immunosuppression and disease severity in sepsis is unknown. The aim of this study was to determine if immunosuppressive bile acids are present in endotoxemia and septic shock and, if so, which patients are particularly at risk. METHODS: To induce experimental endotoxemia in humans, ten healthy volunteers received 2 ng/kg E. coli lipopolysaccharide (LPS). Circulating bile acids were profiled before and after LPS administration. Furthermore, 48 patients with early (shock onset within < 24 h) and severe septic shock (norepinephrine dose > 0.4 µg/kg/min) and 48 healthy age- and sex-matched controls were analyzed for circulating bile acids. To screen for immunosuppressive effects of circulating bile acids, the capability to induce TGR5 activation was computed for each individual bile acid profile by a recently published formula. RESULTS: Although experimental endotoxemia as well as septic shock led to significant increases in total bile acids compared to controls, this increase was mild in most cases. By contrast, there was a marked and significant increase in circulating bile acids in septic shock patients with severe liver failure compared to healthy controls (61.8 µmol/L vs. 2.8 µmol/L, p = 0.0016). Circulating bile acids in these patients were capable to induce immunosuppression, as indicated by a significant increase in TGR5 activation by circulating bile acids (20.4% in severe liver failure vs. 2.8% in healthy controls, p = 0.0139). CONCLUSIONS: Circulating bile acids capable of inducing immunosuppression are present in septic shock patients with severe liver failure. Future studies should examine whether modulation of bile acid metabolism can improve the clinical course and outcome of sepsis in these patients.

Serum Metabolome Signatures Characterizing Co-Infection of Plasmodium falciparum and HBV in Pregnant Women.

Plasmodium falciparum (P. falciparum) and hepatitis B virus (HBV) co-infection is on the rise among pregnant women in northern Ghana. Mono-infection with either of these two pathogens results in unique metabolic alterations. Thus, we aimed to explicate the effects of this co-infection on the metabolome signatures of pregnant women, which would indicate the impacted metabolic pathways and provide useful prognostic or diagnostic markers. Using an MS/MS-based targeted metabolomic approach, we determined the serum metabolome in pregnant women with P. falciparum mono-infection, HBV mono-infection, P. falciparum, and HBV co-infection and in uninfected (control) women. We observed significantly decreased sphingolipid concentrations in subjects with P. falciparum mono-infection, whereas amino acids and phospholipids were decreased in subjects with HBV mono-infection. Co-infections were found to be characterized distinctively by reduced concentrations of phospholipids and hexoses (mostly glucose) as well as altered pathways that contribute to redox homeostasis. Overall, PC ae C40:1 was found to be a good discriminatory metabolite for the co-infection group. PC ae C40:1 can further be explored for use in the diagnosis and treatment of malaria and chronic hepatitis B co-morbidity as well as to distinguish co-infections from cases of mono-infections.

RIPK3 promoter hypermethylation in hepatocytes protects from bile acid-induced inflammation and necroptosis.

Necroptosis facilitates cell death in a controlled manner and is employed by many cell types following injury. It plays a significant role in various liver diseases, albeit the cell-type-specific regulation of necroptosis in the liver and especially hepatocytes, has not yet been conceptualized. We demonstrate that DNA methylation suppresses RIPK3 expression in human hepatocytes and HepG2 cells. In diseases leading to cholestasis, the RIPK3 expression is induced in mice and humans in a cell-type-specific manner. Overexpression of RIPK3 in HepG2 cells leads to RIPK3 activation by phosphorylation and cell death, further modulated by different bile acids. Additionally, bile acids and RIPK3 activation further facilitate JNK phosphorylation, IL-8 expression, and its release. This suggests that hepatocytes suppress RIPK3 expression to protect themselves from necroptosis and cytokine release induced by bile acid and RIPK3. In chronic liver diseases associated with cholestasis, induction of RIPK3 expression may be an early event signaling danger and repair through releasing IL-8.

Metabolomic Profiling in Patients with Heart Failure and Exercise Intolerance: Kynurenine as a Potential Biomarker.

Aims: Metabolic and structural perturbations in skeletal muscle have been found in patients with heart failure (HF) both with preserved (HFpEF) and reduced (HFrEF) ejection fraction in association with reduced muscle endurance (RME). We aimed in the current study to create phenotypes for patients with RME and HFpEF compared to RME HFrEF according to their metabolomic profiles and to test the potential of Kynurenine (Kyn) as a marker for RME. Methods: Altogether, 18 HFrEF, 17 HFpEF, and 20 healthy controls (HC) were prospectively included in the current study. The following tests were performed on all participants: isokinetic muscle function tests, echocardiography, spiroergometry, and varied blood tests. Liquid chromatography tandem mass spectrometry was used to quantify metabolites in serum. Results: Except for aromatic and branched amino acids (AA), patients with HF showed reduced AAs compared to HC. Further perturbations were elevated concentrations of Kyn and acylcarnitines (ACs) in HFpEF and HFrEF patients (p < 0.05). While patients with HFpEF and RME presented with reduced concentrations of ACs (long- and medium-chains), those with HFrEF and RME had distorted AAs metabolism (p < 0.05). With an area under the curve (AUC) of 0.83, Kyn shows potential as a marker in HF and RME (specificity 70%, sensitivity 83%). In a multiple regression model consisting of short-chain-ACs, spermine, ornithine, glutamate, and Kyn, the latest was an independent predictor for RME (95% CI: −13.01, −3.30, B: −8.2 per 1 µM increase, p = 0.001). Conclusions: RME in patients with HFpEF vs. HFrEF proved to have different metabolomic profiles suggesting varied pathophysiology. Kyn might be a promising biomarker for patients with HF and RME.

Effect of therapeutic drug monitoring-based dose optimization of piperacillin/tazobactam on sepsis-related organ dysfunction in patients with sepsis: a randomized controlled trial.

PURPOSE: Insufficient antimicrobial exposure is associated with worse outcomes in sepsis. We evaluated whether therapeutic drug monitoring (TDM)-guided antibiotic therapy improves outcomes. METHODS: Randomized, multicenter, controlled trial from January 2017 to December 2019. Adult patients (n = 254) with sepsis or septic shock were randomly assigned 1:1 to receive continuous infusion of piperacillin/tazobactam with dosing guided by daily TDM of piperacillin or continuous infusion with a fixed dose (13.5 g/24 h if eGFR ≥ 20 mL/min). Target plasma concentration was four times the minimal inhibitory concentration (range ± 20%) of the underlying pathogen, respectively, of Pseudomonas aeruginosa in empiric situation. Primary outcome was the mean of daily total Sequential Organ Failure Assessment (SOFA) score up to day 10. RESULTS: Among 249 evaluable patients (66.3 ± 13.7 years; female, 30.9%), there was no significant difference in mean SOFA score between patients with TDM (7.9 points; 95% CI 7.1-8.7) and without TDM (8.2 points; 95% CI 7.5-9.0) (p = 0.39). Patients with TDM-guided therapy showed a lower 28-day mortality (21.6% vs. 25.8%, RR 0.8, 95% CI 0.5-1.3, p = 0.44) and a higher rate of clinical (OR 1.9; 95% CI 0.5-6.2, p = 0.30) and microbiological cure (OR 2.4; 95% CI 0.7-7.4, p = 0.12), but these differences did not reach statistical significance. Attainment of target concentration was more common in patients with TDM (37.3% vs. 14.6%, OR 4.5, CI 95%, 2.9-6.9, p < 0.001). CONCLUSION: TDM-guided therapy showed no beneficial effect in patients with sepsis and continuous infusion of piperacillin/tazobactam with regard to the mean SOFA score. Larger studies with strategies to ensure optimization of antimicrobial exposure are needed to definitively answer the question.

Transfer of FRozen Encapsulated multi-donor Stool filtrate for active ulcerative Colitis (FRESCO): study protocol for a prospective, multicenter, double-blind, randomized, controlled trial.

BACKGROUND: Ulcerative colitis (UC) is a chronic inflammatory bowel disease with significant morbidity and mortality. Although the precise cause remains unknown, disturbances in the intestinal microbial community have been linked to its pathogenesis. Randomized controlled trials in UC and relapsing Clostridioides difficile infection (CDI) have established fecal microbiota (FM) transfer (FMT) as an effective therapy. In this context, preliminary results indicated that the transfer of sterile fecal microbiota filtrates (<0.2 µm; FMF, FMFT) of donor stool also drives gastrointestinal microbiota changes and eliminates symptoms in CDI patients. However, along with the success of FMT, regulatory agencies issued safety alerts following reports of serious adverse events due to transmission of enteric pathogens through FMT. To reduce this risk, we established an extensive test protocol for our donors and quarantine regulations for the produced capsules, but alternative concepts are desirable. METHODS: Our project is a randomized, controlled, longitudinal, prospective, three-arm, multicenter, double-blind study to determine the safety and efficacy of repeated long-term, multi-donor FM or FMF transfers compared to placebo using oral, frozen capsules in 174 randomized patients with mild to moderate active UC. The primary outcome will be clinical remission at week 12. DISCUSSION: This proposal aims to examine (a) the efficacy of encapsulated transfer of FM and FMF as a therapy for mild to moderate UC, (b) the short- and long-term safety of FMT and FMFT in patients with UC, and (c) the microbial and immunologic changes that occur after FMT and FMFT to help understand how and why it affects inflammatory bowel disease. TRIAL REGISTRATION: ClinicalTrials.gov NCT03843385 . DRKS (Deutsches Register für Klinische Studien) DRKS00020471.

Real-World Effectiveness of Piperacillin/Tazobactam with and without Linezolid for Spontaneous Bacterial Peritonitis.

BACKGROUND: Guidelines recommend empirical therapy with piperacillin/tazobactam (TZP) for spontaneous bacterial peritonitis (SBP) with low risk of multidrug-resistant organisms. Whether coverage of beta-lactam-resistant Gram-positive bacteria, such as ampicillin-resistant Enterococcus faecium, provides clinical benefit in such situations is unknown. METHODS: In this observational study, we investigated the real-world effectiveness of empirical therapy with TZP monotherapy versus TZP plus linezolid (LZD) combination therapy in patients with SBP from two centers. Treatment failure, defined as the need to escalate antibiotic therapy due to in vitro resistance, lack of neutrophil decrease in ascitic fluid, or clinical decision, and 30-day survival were retrospectively assessed. RESULTS: In the first cohort, 100 SBP episodes were empirically treated with TZP + LZD combination therapy (n = 50) or TZP monotherapy (n = 50). Treatment failure was recorded in 48% with TZP monotherapy compared with 16% with TZP + LZD combination therapy (p = 0.001), and this difference persisted after stratification for community-acquired versus hospital-acquired SBP. Although treatment failure after TZP therapy was associated with lower 30-day survival (56% vs. 82%; p = 0.04), 30-day survival with empirical TZP + LZD combination therapy was not different from empirical TZP monotherapy (Kaplan-Meier estimates 74% vs. 69%; p = 0.87). TZP concentrations in ascitic fluid were >32 mg/L in 94% samples after continuous administration. In a second cohort of 41 patients empirically treated with TZP, treatment failure was observed in 37%, which was also higher than in episodes treated with TZP + LZD in cohort 1 (p = 0.03). CONCLUSION: In this retrospective analysis, empirical TZP + LZD combination therapy for SBP was associated with fewer treatment failures without impact on short-term survival.

Introduction of BD Vacutainer® Barricor™ tubes in clinical biobanking and application of amino acid and cytokine quality indicators to Barricor plasma.

OBJECTIVES: The use of BD Vacutainer® Barricor™ tubes (BAR) can reduce turnaround time (TAT) and improve separation of plasma from cellular components using a specific mechanical separator. Concentrations of amino acids (AAs) and cytokines, known to be labile during pre-analytical time delays, were compared in heparin (BAR, BD Heparin standard tube [PST]), EDTA and serum gel tubes (SER) to validate previously identified quality indicators (QIs) in BAR. METHODS: Samples of healthy individuals (n=10) were collected in heparin, EDTA and SER tubes and exposed to varying pre- and post-centrifugation delays at room temperature (RT). Cytokines (interleukin [IL]-8, IL-16 and sCD40L) were analyzed by enzyme-linked immunosorbent assay (ELISA) and AAs were characterized by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). RESULTS: All QIs, AAs/AA ratio and cytokines increased during prolonged blood storage in heparin plasma (PST, BAR) and SER tubes. Comparison of 53 h/1 h pre-centrifugation delay resulted in an increase in taurine (Tau) and glutamic acid (Glu) concentrations by more than three times, soluble CD40L increased by 13.6, 9.2 and 4.3 fold in PST, BAR-CTRL and BAR-FAST, and IL-8 increased even more by 112.8 (PST), 266.1 (BAR-CTRL), 268.1 (BAR-FAST) and 70.0 (SER) fold, respectively. Overall, compared to prolonged blood storage, effects of post-centrifugation delays were less pronounced in all tested materials. CONCLUSIONS: BAR tubes are compatible with the use of several established QIs and can therefore be used in clinical biobanking to reduce pre-analytical TAT without compromising QIs and thus pre-analytical sample quality analysis.

Analysis of Metabolic Markers in Patients with Chronic Heart Failure before and after LVAD Implantation.

Chronic heart failure (HF) is a clinical syndrome characterized by functional impairments of the myocardium. Metabolic and clinical changes develop with disease progression. In an advanced state, left ventricular assist devices (LVADs) are implanted for mechanical unloading. Our study aimed to assess the effects of LVAD implantation on the metabolic phenotypes and their potential to reverse the latter in patients with advanced HF. Plasma metabolites were analyzed by LC-MS/MS in 20 patients with ischemic cardiomyopathy (ICM), 20 patients with dilative cardiomyopathy (DCM), and 20 healthy controls. Samples were collected in HF patients before, 30 days after, and >100 days after LVAD implantation. Out of 188 measured metabolites, 63 were altered in HF. Only three metabolites returned to pre-LVAD concentrations 100 days after LVAD implantation. Pre-LVAD differences between DCM and ICM were mainly observed for amino acids and biogenic amines. This study shows a reversal of metabolite abnormalities in HF as a result of LVAD implantation. The etiology of the underlying disease plays an essential role in defining which specific metabolic parameter is altered in HF and reversed by LVAD implantation. Our findings provide a detailed insight into the disease pattern of ICM and DCM and the potential for reversibility of metabolic abnormalities in HF.

Circulating Bile Acids in Liver Failure Activate TGR5 and Induce Monocyte Dysfunction.

BACKGROUND & AIMS: Retention of bile acids in the blood is a hallmark of liver failure. Recent studies have shown that increased serum bile acid levels correlate with bacterial infection and increased mortality. However, the mechanisms by which circulating bile acids influence patient outcomes still are elusive. METHODS: Serum bile acid profiles in 33 critically ill patients with liver failure and their effects on Takeda G-protein-coupled receptor 5 (TGR5), an immunomodulatory receptor that is highly expressed in monocytes, were analyzed using tandem mass spectrometry, novel highly sensitive TGR5 bioluminescence resonance energy transfer using nanoluciferase (NanoBRET, Promega Corp, Madison, WI) technology, and in vitro assays with human monocytes. RESULTS: Twenty-two patients (67%) had serum bile acids that led to distinct TGR5 activation. These TGR5-activating serum bile acids severely compromised monocyte function. The release of proinflammatory cytokines (eg, tumor necrosis factor α or interleukin 6) in response to bacterial challenge was reduced significantly if monocytes were incubated with TGR5-activating serum bile acids from patients with liver failure. By contrast, serum bile acids from healthy volunteers did not influence cytokine release. Monocytes that did not express TGR5 were protected from the bile acid effects. TGR5-activating serum bile acids were a risk factor for a fatal outcome in patients with liver failure, independent of disease severity. CONCLUSIONS: Depending on their composition and quantity, serum bile acids in liver failure activate TGR5. TGR5 activation leads to monocyte dysfunction and correlates with mortality, independent of disease activity. This indicates an active role of TGR5 in liver failure. Therefore, TGR5 and bile acid metabolism might be promising targets for the treatment of immune dysfunction in liver failure.

Metabolomic profiling of patients with high gradient aortic stenosis undergoing transcatheter aortic valve replacement.

AIM: Aim of our study was to evaluate metabolic changes in patients with aortic stenosis (AS) before and after transcatheter aortic valve replacement (TAVR) and to assess whether this procedure reverses metabolomic alterations. METHODS: 188 plasma metabolites of 30 patients with severe high-gradient aortic valve stenosis (pre-TAVR and 6 weeks post-TAVR) as well as 20 healthy controls (HC) were quantified by liquid chromatography tandem mass spectrometry. Significantly altered metabolites were then correlated to an extensive patient database of clinical parameters at the time of measurement. RESULTS: Out of the determined metabolites, 26.6% (n = 50) were significantly altered in patients with AS pre-TAVR compared to HC. In detail, 5/40 acylcarnitines as well as 10/42 amino acids and biogenic amines were mainly increased in AS, whereas 29/90 glycerophospholipids and 6/15 sphingomyelins were mainly reduced. In the post-TAVR group, 10.1% (n = 19) of metabolites showed significant differences when compared to pre-TAVR. Moreover, we found nine metabolites revealing reversible concentration levels. Correlation with clinically important parameters revealed strong correlations between sphingomyelins and cholesterol (r = 0.847), acylcarnitines and brain natriuretic peptide (r = 0.664) and showed correlation of acylcarnitine with an improvement of left ventricular (LV) ejection fraction (r = - 0.513) and phosphatidylcholines with an improvement of LV mass (r = - 0.637). CONCLUSION: Metabolic profiling identified significant and reversible changes in circulating metabolites of patients with AS. The correlation of circulating metabolites with clinical parameters supports the use of these data to identify novel diagnostic as well as prognostic markers for disease screening, pathophysiological studies as well as patient surveillance.

Targeting Complement C5a Receptor 1 for the Treatment of Immunosuppression in Sepsis.

Complement factor C5a was originally identified as a powerful promoter of inflammation through activation of the C5a receptor 1 (C5ar1). Recent evidence suggests involvement of C5a not only in pro- but also in anti-inflammatory signaling. The present study aims to unveil the role of C5ar1 as potential therapeutic target in a murine sepsis model. Our study discloses a significantly increased survival in models of mild to moderate but not severe sepsis of C5ar1-deficient mice. The decreased mortality of C5ar1-deficient mice is accompanied by improved pathogen clearance and largely preserved liver function. C5ar1-deficient mice exhibited a significantly increased production of the pro-inflammatory mediator interferon-γ (IFN-γ) and a decreased production of the anti-inflammatory cytokine interleukin-10 (IL-10). Together, these data uncover C5a signaling as a mediator of immunosuppressive processes during sepsis and describe the C5ar1 and related changes of the IFN-γ to IL-10 ratio as markers for the immunological (dys)function accompanying sepsis.

Lipid metabolic signatures deviate in sepsis survivors compared to non-survivors.

Sepsis remains a major cause of death despite advances in medical care. Metabolic deregulation is an important component of the survival process. Metabolomic analysis allows profiling of critical metabolic functions with the potential to classify patient outcome. Our prospective longitudinal characterization of 33 septic and non-septic critically ill patients showed that deviations, independent of direction, in plasma levels of lipid metabolites were associated with sepsis mortality. We identified a coupling of metabolic signatures between liver and plasma of a rat sepsis model that allowed us to apply a human kinetic model of mitochondrial beta-oxidation to reveal differing enzyme concentrations for medium/short-chain hydroxyacyl-CoA dehydrogenase (elevated in survivors) and crotonase (elevated in non-survivors). These data suggest a need to monitor cellular energy metabolism beyond the available biomarkers. A loss of metabolic adaptation appears to be reflected by an inability to maintain cellular (fatty acid) metabolism within a "corridor of safety".

Association of proteome and metabolome signatures with severity in patients with community-acquired pneumonia.

A combined OMICS screening approach of human plasma and serum was used to characterize protein and metabolome signatures displaying association to severity of Community-acquired pneumonia (CAP). 240 serum and BD P100 EDTA plasma samples from patients diagnosed with CAP, collected during the day of enrolment to the hospital, were analyzed by a metabolomic and proteomic approach, respectively. Disease severity of CAP patients was stratified using the Sequential Organ Failure Assessment (SOFA) score. Quantitative proteome and metabolome data, derived by LC-MS/MS, were associated to SOFA and specific parameters of SOFA using linear regression models adjusted for age, BMI, sex, smoking and technical variables. Both proteome and metabolome profiling revealed remarkable strong changes in plasma and serum composition in relation to severity of CAP. Proteins and metabolites displaying SOFA associated levels are involved in immune response, particularly in processes of lipid metabolism. Proteins, which show an association to SOFA score, are involved in acute phase response, coagulation, complement activation and inflammation. Many of these metabolites and proteins displayed not only associations to SOFA, but also to parameters of SOFA score, which likely reflect the strong influence of lung-, liver-, kidney- and heart-dysfunction on the metabolome and proteome patterns. SIGNIFICANCE: Community-acquired pneumonia is the most frequent infection disease with high morbidity and mortality. So far, only few studies focused on the identification of proteins or metabolites associated to severity of CAP, often based on smaller sample sets. A screening for new diagnostic markers requires extensive sample collections in combination with high quality clinical data. To characterize the proteomic and metabolomics pattern associated to severity of CAP we performed a combined metabolomics and proteomic approach of serum and plasma sample from a multi-center clinical study focused on patients with CAP, requiring hospitalization. The results of this association study of omics data to the SOFA score enable not only an interpretation of changes in molecular patterns with severity of CAP but also an assignment of altered molecules to dysfunctions of respiratory, renal, coagulation, cardiovascular systems as well as liver.

Quality Assessment of the Preanalytical Workflow in Liquid Biobanking: Taurine as a Serum-Specific Quality Indicator for Preanalytical Process Variations.

The scientific impact of translational biomedical research largely depends on the availability of high-quality biomaterials. However, evidence-based and robust quality indicators (QIs) covering the most relevant preanalytical variations are still lacking. The aim of this study was to identify and validate a QI suitable for assessing time-to-centrifugation (TTC) delays in human liquid biospecimens originating from both healthy and diseased individuals. Serum and plasma samples with varying TTCs were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in a pilot cohort of healthy individuals to identify a suitable QI candidate. Taurine (TAU), as a TTC QI candidate, was validated in healthy individuals and patients with rheumatologic and cardiologic diseases, considering the (1) preanalytical handling temperature, (2) platelet count, and (3) postcentrifugation delay. For discrimination of high TTC (TTC >60 minutes) from low TTC serum specimens, a probability calculation tool was developed (Triple-T-cutoff-model). TTC-dependent changes in healthy individuals were observed for amino acids, particularly TAU. Validation of the TAU levels in an independent cohort of healthy individuals revealed a time-dependent increase in serum, but not in plasma, for a TTC delay of 30-240 minutes. TAU increases were dependent on the handling temperature and platelet count and volume. By contrast, no changes in TAU concentrations were observed for additional postcentrifugation delays. Validation of TAU and the Triple-T-cutoff-model, in rheumatologic/cardiologic patient collectives, allowed the discrimination of samples with TTC ≤60 min/>60 min with estimated AUROC (area under the receiver operating characteristic curve) values of 89% [78%-100%]/86% [71%-100%] and 91% [79%-100%]/84% [68%-100%], respectively. Considering the preanalytical handling temperature and platelet count and volume, TAU and the Triple-T-cutoff-model represent reliable QIs for TTC >60 minutes in serum samples from healthy individuals and selected rheumatologic/cardiologic patients. However, further studies in larger patient collectives with various diseases are needed to assess the robustness and potential of the QIs presented in this article as biobanking quality assurance/quality control tools to support high-quality biomedical research.

Simultaneous Quantification of Nine Antimicrobials by LC-MS/MS for Therapeutic Drug Monitoring in Critically Ill Patients.

BACKGROUND: Adequate antibiotic treatment is a prerequisite for the successful treatment of systemic infections. Based on accumulating scientific evidence, a fixed dosage regimen can lead to insufficient and ineffective antibiotic therapy. Thus, the aim of this study was to develop and validate a simplified, but sensitive method for the simultaneous quantification of antimicrobials by using liquid chromatography with tandem mass spectrometry (LC-MS/MS) for the development of personalized therapy regimens using therapeutic drug monitoring. METHODS: A method was developed for the simultaneous quantification of 9 antimicrobials (aciclovir, ampicillin, cefuroxime, ciprofloxacin, meropenem, metronidazole, piperacillin, rifampicin, and tazobactam) in lithium-heparin plasma. A simple sample preparation method and a chromatographic run time of 10 minutes enabled the quick processing of the samples. The method was validated according to the guidelines for bioanalytical method validation of the European Medicines Agency and addressed sensitivity, specificity, linearity, accuracy, precision, dilution integrity, carry-over, recovery, matrix effects, and stability. RESULTS: The chromatographic run time was 10 minutes and antimicrobials eluted at retention times ranging from 1.1 to 2.2 minutes. Calibration curve for all antimicrobials was linear over a range of 1-100 mg/L, and a 2-fold or 5-fold dilution of the samples was possible. The method accuracy ranged from 85.1% to 114.9% for all measured antimicrobials, and the within- and between-run precision values were <11.9% and <16.5% for the lower limit of quantification. No interferences and carry-over were observed. The samples were stable for at least 5 hours at room temperature or in the autosampler (10°C). CONCLUSIONS: The LC-MS/MS method developed in this study is appropriate and practical for the therapeutic drug monitoring of antimicrobials in the daily clinical laboratory practice because of its short analysis time, the need for a small amount of plasma, high specificity, and accuracy.