Objectives: The analysis of organic acids in urine is an important part of the diagnosis of inherited metabolic disorders (IMDs), for which gas chromatography coupled with mass spectrometry is still predominantly used. Methods: Ultra-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for urinary organic acids, acylcarnitines and acylglycines was developed and validated. Sample preparation consists only of dilution and the addition of internal standards. Raw data processing is quick and easy using selective scheduled multiple reaction monitoring mode. A robust standardised value calculation as a data transformation together with advanced automatic visualisation tools are applied for easy evaluation of complex data. Results: The developed method covers 146 biomarkers consisting of organic acids (n=99), acylglycines (n=15) and acylcarnitines (n=32) including all clinically important isomeric compounds present. Linearity with r(2)>0.98 for 118 analytes, inter-day accuracy between 80 and 120 % and imprecision under 15 % for 120 analytes were achieved. Over 2 years, more than 800 urine samples from children tested for IMDs were analysed. The workflow was evaluated on 93 patient samples and ERNDIM External Quality Assurance samples involving a total of 34 different IMDs. Conclusions: The established LC-MS/MS workflow offers a comprehensive analysis of a wide range of organic acids, acylcarnitines and acylglycines in urine to perform effective, rapid and sensitive semi-automated diagnosis of more than 80 IMDs.

BACKGROUND/AIM:Biomarkers that would identify patients unlikely to respond to immunotherapy with immune checkpoint inhibitors (ICIs) remain an unmet medical need.PATIENTS AND METHODS:In the present study, we have retrospectively evaluated the association between biomarkers of immune activation and outcome in metastatic renal cell carcinoma (mRCC) patients treated with ICIs. The laboratory and clinical data of 79 consecutive patients with histologically confirmed mRCC treated with ICI-based immunotherapy have been analyzed.RESULTS:Patients who progressed or died at 4 months had higher prognostic score, higher serum C-reactive protein (CRP) and neopterin, and urinary neopterin, and lower serum albumin and hemoglobin concentration.CONCLUSION:Biomarkers of activation of immune response, in particular serum neopterin/creatinine ratio, are associated with outcome in mRCC patients treated with ICI immunotherapy.

Newborn screening (NBS) of inborn errors of metabolism (IEMs) is based on the reference ranges established on a healthy newborn population using quantile statistics of molar concentrations of biomarkers and their ratios. The aim of this paper is to investigate whether multivariate independent component analysis (ICA) is a useful tool for the analysis of NBS data, and also to address the structure of the calculated ICA scores. NBS data were obtained from a routine NBS program performed between 2013 and 2022. ICA was tested on 10,213/150 free-diseased controls and 77/20 patients (9/3 different IEMs) in the discovery/validation phases, respectively. The same model computed during the discovery phase was used in the validation phase to confirm its validity. The plots of ICA scores were constructed, and the results were evaluated based on 5sd levels. Patient samples from 7/3 different diseases were clearly identified as 5sd-outlying from control groups in both phases of the study. Two IEMs containing only one patient each were separated at the 3sd level in the discovery phase. Moreover, in one latent variable, the effect of neonatal birth weight was evident. The results strongly suggest that ICA, together with an interpretation derived from values of the "average member of the score structure", is generally applicable and has the potential to be included in the decision process in the NBS program.

Three genetically determined enzyme defects of purine de novo synthesis (PDNS) have been identified so far in humans: adenylosuccinate lyase (ADSL) deficiency, 5-amino-4-imidazole carboxamide-ribosiduria (AICA-ribosiduria), and deficiency in bifunctional enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthase (PAICS). Clinical signs of these defects are mainly neurological, such as seizures, psychomotor retardation, epilepsy, autistic features, etc. This work aims to describe the metabolic changes of CRISPR-Cas9 genome-edited HeLa cells deficient in the individual steps of PDNS to better understand known and potential defects of the pathway in humans. High-performance liquid chromatography coupled with mass spectrometry was used for both targeted and untargeted metabolomic analyses. The statistically significant features from the untargeted study were identified by fragmentation analysis. Data from the targeted analysis were processed in Cytoscape software to visualize the most affected metabolic pathways. Statistical significance of PDNS intermediates preceding deficient enzymes was the highest (p-values 10 × 10−7–10 × 10−15) in comparison with the metabolites from other pathways (p-values of up to 10 × 10−7). Disturbed PDNS resulted in an altered pool of adenine and guanine nucleotides. However, the adenylate energy charge was not different from controls. Different profiles of acylcarnitines observed among deficient cell lines might be associated with a specific enzyme deficiency rather than global changes related to the PDNS pathway. Changes detected in one-carbon metabolism might reduce the methylation activity of the deficient cells, thus affecting the modification state of DNA, RNA, and proteins.

Long chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD/MTPD) and medium chain acyl-CoA dehydrogenase deficiency (MCADD) were included in the expanded neonatal screening program (ENBS) in Czechia in 2009, allowing for the presymptomatic diagnosis and nutritional management of these patients. The aim of our study was to assess the nationwide impact of ENBS on clinical outcome. This retrospective study analysed acute events and chronic complications and their severity in pre-ENBS and post-ENBS cohorts. In total, 28 children (12 before, 16 after ENBS) were diagnosed with LCHADD/MTPD (incidence 0.8/100,000 before and 1.2/100,000 after ENBS). In the subgroup detected by ENBS, a significantly longer interval from birth to first acute encephalopathy was observed. In addition, improvement in neuropathy and cardiomyopathy (although statistically non-significant) was demonstrated in the post-ENBS subgroup. In the MCADD cohort, we included 69 patients (15 before, 54 after ENBS). The estimated incidence rose from 0.7/100,000 before to 4.3/100,000 after ENBS. We confirmed a significant decrease in the number of episodes of acute encephalopathy and lower proportion of intellectual disability after ENBS (p < 0.0001). The genotype–phenotype correlations suggest a new association between homozygosity for the c.1528C > G variant and more severe heart involvement in LCHADD patients.

3‐Hydroxy‐3‐methylglutaryl‐coenzyme A lyase deficiency (HMGCLD) is a rare autosomal recessively inherited metabolic disorder. Patients suffer from avoidable neurologically devastating metabolic decompensations and thus would benefit from newborn screening (NBS). The diagnosis is currently made by measuring dry blood spot acylcarnitines (C5OH and C6DC) followed by urinary organic acid profiling for the differential diagnosis from several other disorders. Using untargeted metabolomics (reversed‐phase UHPLC coupled to an Orbitrap Elite hybrid mass spectrometer) of plasma samples from 5 HMGCLD patients and 19 age‐matched controls, we found 3‐methylglutaconic acid and 3‐hydroxy‐3‐methylglutaric acid, together with 3‐hydroxyisovalerylcarnitine as the most discriminating metabolites between the groups. In order to evaluate the NBS potential of these metabolites we quantified the most discriminating metabolites from untargeted metabolomics in 23 blood spots from 4 HMGCLD patients and 55 controls by UHPLC tandem mass spectrometry. The results provide a tool for expanded NBS of HMGCLD using tandem mass spectrometry. Selected reaction monitoring transition 262/85 could be used in a first‐tier NBS analysis to screen for elevated 3‐hydroxyisovalerylcarnitine. In a positive case, a second‐tier analysis of 3‐hydroxy‐3‐methylglutaric acid and 3‐methylglutaconic acid in a dry blood spot using UHPLC tandem mass spectrometry instruments confirms the diagnosis. In conclusion, we describe the identification of new diagnostic biomarkers for HMGCLD and their application in NBS in dry blood spots. By using second‐tier testing, all patients with HMGCLD were unequivocally and correctly diagnosed.

aSummary: Untargeted liquid chromatography-high-resolution mass spectrometry analysis produces a large number of features which correspond to the potential compounds in the sample that is analyzed. During the data processing, it is necessary to merge features associated with one compound to prevent multiplicities in the data and possible misidentification. The processing tools that are currently employed use complex algorithms to detect abundances, such as adducts or isotopes. However, most of them are not able to deal with unpredictable adducts and in-source fragments. We introduce a simple open-source R-script CROP based on Pearson pairwise correlations and retention time together with a graphical representation of the correlation network to remove these redundant features.

Clinical metabolomics aims at finding statistically significant differences in metabolic statuses of patient and control groups with the intention of understanding pathobiochemical processes and identification of clinically useful biomarkers of particular diseases. After the raw measurements are integrated and pre-processed as intensities of chromatographic peaks, the differences between controls and patients are evaluated by both univariate and multivariate statistical methods. The traditional univariate approach relies on t-tests (or their nonparametric alternatives) and the results from multiple testing are misleadingly compared merely by p-values using the so-called volcano plot. This paper proposes a Bayesian counterpart to the widespread univariate analysis, taking into account the compositional character of a metabolome. Since each metabolome is a collection of some small-molecule metabolites in a biological material, the relative structure of metabolomic data, which is inherently contained in ratios between metabolites, is of the main interest. Therefore, a proper choice of logratio coordinates is an essential step for any statistical analysis of such data. In addition, a concept of b-values is introduced together with a Bayesian version of the volcano plot incorporating distance levels of the posterior highest density intervals from zero. The theoretical background of the contribution is illustrated using two data sets containing samples of patients suffering from 3-hydroxy-3-methylglutaryl-CoA lyase deficiency and medium-chain acyl-CoA dehydrogenase deficiency. To evaluate the stability of the proposed method as well as the benefits of the compositional approach, two simulations designed to mimic a loss of samples and a systematical measurement error, respectively, are added.

Objectives: Presymptomatic detection of patients with rare diseases (RD), defined by a population frequency less than 1 : 2,000, is the task of newborn screening (NBS). In the Czech Republic (CZ), currently eighteen RD are screened: phenylketonuria/hyperphenylalaninemia (PKU/HPA), congenital hypothyroidism (CH), congenital adrenal hyperplasia (CAH), cystic fibrosis (CF), medium chain acyl-CoA dehydrogenase deficiency (MCADD), long chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), very long chain acyl-CoA dehydrogenase deficiency (VLCADD), carnitine palmitoyl transferase I and II deficiency (CPTID, CPTIID), carnitine-acylcarnitine translocase deficiency (CACTD), maple syrup urine disease (MSUD), glutaric aciduria type I (GA I), isovaleryl-CoA dehydrogenase deficiency (IVA), argininemia (ARG), citrullinemia (CIT), biotinidase deficiency (BTD), cystathionine beta-synthase-deficient homocystinuria (CBSD HCU), and methylenetetrahydrofolate reductase deficiency homocystinuria (MTHFRD HCU). The aim was to analyze the prevalence of RD screened by NBS in CZ. Methods: We examined the NBS programme in CZ from 1 January 2010 to 31 December 2017, which covered 888,891 neonates. Dried blood spots were primarily analyzed using fluorescence immuno-assay, tandem mass spectrometry and fluorimetry. Results: The overall prevalence of RD among the neonate cohort was 1 : 1,043. Individually, 1 : 2,877 for CH, 1 : 5,521 for PKU/HPA, 1 : 6,536 for CF (1 : 5,887 including false negative patients), 1 : 12,520 for CAH, 1 : 22,222 for MCADD, 1 : 80,808 for LCHADD, 1 : 177,778 for GA I, 1 : 177,778 for IVA, 1 : 222,223 for VLCADD, 1 : 296,297 for MSUD, 1 : 8,638 for BTD, and 1 : 181,396 for CBSD HCU. Conclusions: The observed prevalence of RD, based on NBS, corresponds to that expected, more precisely it was higher for BTD and lower for MSUD, IVA, CBSD HCU, MCADD and VLCADD. Early detection of rare diseases by means of NBS is an effective secondary prevention tool.

Urea, as an end product of protein metabolism and an abundant polar compound, significantly complicates the metabolomic analysis of urine by GC-MS. We developed a sample preparation method removing urea from urine samples prior the GC-MS analysis. The method based on urease immobilized on magnetic microparticles was compared with the others that are conventionally used (liquid-liquid extraction, free urease protocol), and samples without any treatment. To study the impact of sample preparation approaches on the quality of analytical data, we employed comprehensive metabolomic analysis (using both GC-MS and LC-MS/MS platforms) of standard material based on human urine. Multivariate statistical analysis has shown that immobilized urease treatment provides similar results to a free urease approach. However, significant alterations in the profiles of metabolites were observed in the samples without any treatment and after the extraction. Compared to other approaches that were tested, the immobilization of urease on microparticles reduces both the number of artifacts and the variability of the metabolites (average CV of extraction 19.7%, no treatment 11.4%, free urease 5.0%, and immobilized urease 2.5%). The method that was developed was applied in a GC-MS metabolomic experiment of glutaric aciduria type I, where both known diagnostically important biomarkers and unknowns, as the most discriminating compounds, were found.

Orthogonality is a key parameter in the evaluation of the performance of a 2D chromatography-based separation system. Two different perspectives on orthogonality are determined: the extent of the separation space utilized (global orthogonality) and the uniformity of the coverage of the separation space (local orthogonality). This work aims to elucidate the impact of sample dimensionality (the number of separation processes involved) on orthogonality evaluation through the use of descriptors from seven different algorithms utilizing mutually different properties of a chromatogram: Pearson correlation, conditional entropy, asterisk equations, convex hull, arithmetic mean (AN) and harmonic mean of the nearest neighbor, and geometric surface coverage (SC). Artificial chromatograms generated in silico and real GC × GC separations of diesel, plasma, and urine were used for the evaluation of orthogonality. The sample dimensionality has a deep effect on the orthogonality results of all approaches. The SC algorithm emerged as the best descriptor of local orthogonality samples of both low and high dimensionality, the AN algorithm on the global orthogonality of low-dimensionality samples. However, in the case of samples of high dimensionality, AN consistently indicated just the exploitation of the whole separation space; therefore, only local orthogonality is optimized by means of SC. Since no approach was able to monitor both global and local orthogonality as a single value, a new descriptor, ASCA, was developed. It combines the best global (AN) and local (SC) orthogonality algorithms by averaging, giving the same importance to data spread and crowding. ASCA thus provides the best estimation of orthogonality.

Data outliers can carry very valuable information and might be most informative for the interpretation. Nevertheless, they are often neglected. An algorithm called cellwise outlier diagnostics using robust pairwise log ratios (cell-rPLR) for the identification of outliers in single cell of a data matrix is proposed. The algorithm is designed for metabolomic data, where due to the size effect, the measured values are not directly comparable. Pairwise log ratios between the variable values form the elemental information for the algorithm, and the aggregation of appropriate outlyingness values results in outlyingness information. A further feature of cell-rPLR is that it is useful for biomarker identification, particularly in the presence of cellwise outliers. Real data examples and simulation studies underline the good performance of this algorithm in comparison with alternative methods.

Abstract Background The prediction of outcome in comatose patients after out of hospital cardiac arrest (OHCA) has major ethical and socioeconomic implications. At present, there is a lack of data comparing the predictive value from cardiac arrest localization to hospital distance in OHCA survivors treated with endovascular therapeutic hypothermia. Methods 86 patients (64±14 years, 69 men) were evaluated after OHCA due to ventricular fibrillation (VF) during an acute myocardial infarction (MI). All patients (NSTEMI 28%, STEMI 72%) were indicated for urgent coronary angiography, echocardiography for left ventricular ejection fraction (LVEF) estimation using Simpson biplane formula and treated with mild therapeutic hypothermia (MTH) using intravascular temperature management to maintain target temperature (33 °C) for 24 hours. The Cerebral Performance Categories scale (CPC) was used as the outcome measure and was assessed 3 months post admission; a CPC of 3–5 was regarded as a poor outcome (n=45), and a CPC of 1–2 (n=41) as a good outcome. Results Distance to hospital was significantly higher (p=0.0473) in patients with poor outcomes (CPC 3–5) after OHCA (37.5±4.5 km) compared with CPC 1–2 patients (27.1±4.4 km). No significant differences in return of spontaneous circulation time (21; 10.5–47.5 95th percentile vs. 23; 10.0–50.0, p=0.738), lactate (7.8; 4.5–12.4 vs. 8.4; 5.4–13.5, p=0.54), LVEF (40; 22–50 vs. 40; 21–62%, p=0.208), peak cardiac troponin T (1.5; 0.08–10.00 vs. 0.64; 0.04–5.28 μg/L, p=0.078), NSE (29.2; 15.7–54.9 vs. 25.8; 13.6–52.3 μg/L, p=0.26) and S100-B (0.17; 0.09–1.69 vs. 0.19; 0.04–1.14 μg/L, p=0.734) were found in CPC 3–5 and CPC 1–2 groups comparison. Using an optimal cut-off value ≥33 km calculated from the receiver operating characteristic curve (area under curve = 0.62; p=0.004), the sensitivity of predicting survival with poor neurological outcome was 61% and the specificity was 62%. Conclusions In patients after OHCA for VF during MI, distance from cardiac arrest localization to hospital gives reliable and on return of spontaneous circulation time independent prognostic information concerning outcome after cardiopulmonary resuscitation. Acknowledgement/Funding Grant support FNOL RIV 87-85

S28463 (S28), a ligand for Toll-like receptor 7/8, has been shown to have antiinflammatory properties in rodent models of allergic asthma. The principle goal of this study was to assess whether these antiinflammatory effects can also be observed in a nonhuman primate (NHP) model of allergic asthma. NHPs were sensitized then challenged with natural allergen, Ascaris suum extract. The animals were treated with S28 orally before each allergen challenge. The protective effect of S28 in NHPs was assessed by measuring various asthma-related phenotypes. We also characterized the metabolomic and proteomic signatures of the lung environment and plasma to identify markers associated with the disease and treatment. Our data demonstrate that clinically relevant parameters, such as wheal and flare response, blood IgE levels, recruitment of white blood cells to the bronchoalveolar space, and lung responsiveness, are decreased in the S28-treated allergic NHPs compared with nontreated allergic NHPs. Furthermore, we also identified markers that can distinguish allergic from nonallergic or allergic and drug-treated NHPs, such as metabolites, phosphocreatine and glutathione, in the plasma and BAL fluid, respectively; and inflammatory cytokines, IL-5 and IL-13, in the bronchoalveolar lavage fluid. Our preclinical study demonstrates that S28 has potential as a treatment for allergic asthma in primate species closely related to humans. Combined with our previous findings, we demonstrate that S28 is effective in different models of asthma and in different species, and has the antiinflammatory properties clinically relevant for the treatment of allergic asthma.

To compare preoperative intestinal trefoil factor 3 (TFF3), allograft inflammatory factor-1 (AIF-1) and calgizzarin (S100-A11) serum levels in patients with endometrial cancer, endometrial hyperplasia and in healthy female controls. Serum levels of TFF3, S100- A11 and AIP-1 were analyzed in 98 consecutive patients with histologically verified endometrial cancer, in 43 patients with endometrial hyperplasia diagnosed during hysteroscopy and 24 controls with benign disease. Results were correlated with urinary neopterin/creatinine ratio, serum kynurenine, tryptophan, retinol, alpha-tocopherol, vitamin D, citrulline, C-reactive protein, interleukin-6 and clinical characteristics. S100-A11, and AIF-1 levels were higher in endometrial hyperplasia patients than in controls, and also significantly higher in endometrial cancer than in patients with endometrial hyperplasia. Serum concentrations of TFF3 and S100-A11 were associated with tumor stage and lymph node status. TFF3 exhibited positive correlation with age, IL-6, vitamin D, kynurenine, urinary neopterin/creatinine ratio and kynurenine/tryptophan ratio. S100-A11, as well as AIF-1 correlated positively with Il-6 and TFF3. TFF3, S100-A11 and AIF-1 represent potential biomarkers in patients with endometrial cancer. TFF3 and S100-A11 increase with tumor stage and lymph node involvement, reflecting higher tumor mass that is also associated with increased concentration of biomarkers of immune dysfunction.

Introduction: The aim of prospective study was to evaluate the ability of echocardiography and cardiac biomarkers to predict in-hospital mortality and the risk of brain infarction during a 12-month follow-up period (FUP) with anticoagulation in pulmonary embolism (PE) patients. Methods: Eighty-eight consecutive acute PE patients (39 men, mean age 63 years) were enrolled; 78 underwent baseline echocardiography and brain magnetic resonance imaging (MRI). After a 12-month FUP, 58 underwent brain MRI. In-hospital mortality and the rates of new ischemic brain lesions (IBL) on MRI with clinical ischemic stroke (IS) events were predicted based on echocardiography (patent foramen ovale presence with right-to-left shunt - PFO/RLS; right/left ventricle diameter ratio - RV/LD; tricuspid annulus plane systolic excursion - TAPSE; tricuspid annulus systolic velocity - S-T; pulmonary artery systolic pressure - PASP) and biomarkers results (amino-terminal fragment of brain natriuretic peptide - NT-proBNP and cardiac troponin T - cTnT). Results: Our series involved 88 patients, of whom 11 (12.5%) presented high-risk PE, 24 (27.3%) intermediate-high risk PE, 19 (21.6%) intermediate-low risk PE and 34 (38.6%) patients had low risk PE. Nine patients (10.2%) died during hospitalization including high-risk PE [6/9 (66.6%)] and intermediate-high-risk PE [3/24 (12.5%)]. cTnT [odds ratio (OR) 4.3; 95% confidence interval 0.59-31.3, P = 0.014], NT-proBNP (OR 14.2 [1.5-133.4], P = 0.02), RV/LD = >= 0.79 (OR 36.6 [4.2316.4], P = 0.001), TAPSE (OR 0.55 [0.34-0.92, P = 0.022) and PASP = >= 51.5 mmHg (OR 33.3 [3.8-292.6], P = 0.022) were predictors of in-hospital mortality. Seventeen patients (19.3%) experienced IS (n = 8) or new IBL (n = 9). On multivariate analysis, PFO/RLS (OR 27.1 [3.0-245.3], P = 0.003) and S-T = <= 14.5 cm/s (OR 34.1 [CI 3.4-344.0], P = 0.003) were independent predictors of IS and IBL risk. Conclusions: High blood troponin T, NT-proBNP, RV dilatation/systolic dysfunction and pulmonary hypertension predicted in-hospital mortality. PFO/RLS presence and S-T were predictors of clinically apparent/silent brain infarction. (c) 2017 The Czech Society of Cardiology. Published by Elsevier Sp. z o. o. All rights reserved.

Purines are essential molecules for all forms of life. In addition to constituting a backbone of DNA and RNA, purines play roles in many metabolic pathways, such as energy utilization, regulation of enzyme activity, and cell signaling. The supply of purines is provided by two pathways: the salvage pathway and de novo synthesis. Although purine de novo synthesis (PDNS) activity varies during the cell cycle, this pathway represents an important source of purines, especially for rapidly dividing cells. A method for the detailed study of PDNS is lacking for analytical reasons (sensitivity) and because of the commercial unavailability of the compounds. The aim was to fully describe the mass spectrometric fragmentation behavior of newly synthesized PDNS-related metabolites and develop an analytical method. Except for four initial ribotide PDNS intermediates that preferentially lost water or phosphate or cleaved the forming base of the purine ring, all the other metabolites studied cleaved the glycosidic bond in the first fragmentation stage. Fragmentation was possible in the third to sixth stages. A liquid chromatography-high-resolution mass spectrometric method was developed and applied in the analysis of CRISPR-Cas9 genome-edited HeLa cells deficient in the individual enzymatic steps of PDNS and the salvage pathway. The identities of the newly synthesized intermediates of PDNS were confirmed by comparing the fragmentation patterns of the synthesized metabolites with those produced by cells (formed under pathological conditions of known and theoretically possible defects of PDNS). The use of stable isotope incorporation allowed the confirmation of fragmentation mechanisms and provided data for future fluxomic experiments. This method may find uses in the diagnosis of PDNS disorders, the investigation of purinosome formation, cancer research, enzyme inhibition studies, and other applications.

The case of atypical myopathy (AM) in newborn Haflinger foal with clinical signs of depression and weakness appearing 6 hours after birth resulting in recumbency 12 hours after birth is described. The foal's dam was diagnosed with AM in the 6th month of gestation based on clinical signs of a myopathy, elevated serum activity of creatine kinase, metabolomic analysis and the presence of methylenecyclopropyl acetyl carnitine (MCPA-carnitine) in the blood. At the time of delivery, the mare was grazing on a pasture near sycamore trees but was free of clinical signs of AM. Metabolomic analysis of the foal's blood revealed increased concentrations of acylcarnitines and MCPA-carnitine consistent with metabolic profiles of blood from AM affected horses. Two theories could explain this observation (a) hypoglycin A or its metabolites accumulated in the mare's placenta with consequent transfer to fetus or (b) these compounds were secreted into mare's milk.