Dynamics of serum levels and reference ranges of copeptin in the 3rd trimester of pregnancy in healthy pregnant women with uncomplicated pregnancy and delivery.

OBJECTIVE: Copeptin is a stable fragment of vasopressin. Copeptin levels have been found to reflect the degree of endothelial stress in various conditions, including acute coronary syndrome. Copeptin may be a bio marker for endothelial stress during pregnancy. However, there is still a lack of understanding of its dynamics and levels throughout pregnancy. This study aims to describe intra-individual and longitudinal changes in copeptin levels at 30th and 36th gestational weeks in healthy pregnant women with uncomplicated pregnancy and delivery and to establish specific reference ranges. METHODS: A total of 125 pregnant women with uncomplicated pregnancy and delivery were included. These women were monitored throughout their pregnancy and gave birth at the Department of Obstetrics and Gynecology Olomouc University Hospital. The blood was taken at ~30 and ~36 gestational weeks. Serum copeptin levels were measured using a Kryptor Compact PLUS analyzer. For statistics, we used R software and the "referenceRanges" package. RESULTS: It was found that serum levels of copeptin were significantly higher in the 36th week group than in the 30th week group (P < 0.05). Cook's distance was used to eliminate outliers. The 30th week median was 3.377 pmol/l, reference range = 1.343-7.829 pmol/l, and the 36 week was median 4.735 pmol/l and reference range = 2.06-13.2 pmol/l. In the 36th week reference range, the median was higher than in healthy, non-pregnant women (P < 0.05). Copeptin values can exceed 10 pmol/l, particularly after the 36th week. In the 3rd trimester, this value may indicate cardiovascular and endothelial overload. CONCLUSION: Copeptin levels were found to vary significantly depending on gestational week. The proposed reference ranges take into account the increased secretion of vasopressin in pregnancy. The existence of specific upper reference limits represents a potential advantage in detecting pregnant women prone to hypertensive disease in the 3rd trimester.

Multivariate Independent Component Analysis Identifies Patients in Newborn Screening Equally to Adjusted Reference Ranges.

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.

Impact of sample dimensionality on orthogonality metrics in comprehensive two-dimensional separations.

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.

Metabolic status of CSF distinguishes rats with tauopathy from controls.

BACKGROUND: Tauopathies represent heterogeneous groups of neurodegenerative diseases that are characterised by abnormal deposition of the microtubule-associated protein tau. Alzheimer's disease is the most prevalent tauopathy, affecting more than 35 million people worldwide. In this study we investigated changes in metabolic pathways associated with tau-induced neurodegeneration. METHODS: Cerebrospinal fluid (CSF), plasma and brain tissue were collected from a transgenic rat model for tauopathies and from age-matched control animals. The samples were analysed by targeted and untargeted metabolomic methods using high-performance liquid chromatography coupled to mass spectrometry. Unsupervised and supervised statistical analysis revealed biochemical changes associated with the tauopathy process. RESULTS: Energy deprivation and potentially neural apoptosis were reflected in increased purine nucleotide catabolism and decreased levels of citric acid cycle intermediates and glucose. However, in CSF, increased levels of citrate and aconitate that can be attributed to glial activation were observed. Other significant changes were found in arginine and phosphatidylcholine metabolism. CONCLUSIONS: Despite an enormous effort invested in development of biomarkers for tauopathies during the last 20 years, there is no clinically used biomarker or assay on the market. One of the most promising strategies is to create a panel of markers (e.g., small molecules, proteins) that will be continuously monitored and correlated with patients' clinical outcome. In this study, we identified several metabolic changes that are affected during the tauopathy process and may be considered as potential markers of tauopathies in humans.

Sample-independent approach to normalize two-dimensional data for orthogonality evaluation using whole separation space scaling.

Orthogonality is a key parameter that is used to evaluate the separation power of chromatography-based two-dimensional systems. It is necessary to scale the separation data before the assessment of the orthogonality. Current scaling approaches are sample-dependent, and the extent of the retention space that is converted into a normalized retention space is set according to the retention times of the first and last analytes contained in a unique sample to elute. The presence or absence of a highly retained analyte in a sample can thus significantly influence the amount of information (in terms of the total amount of separation space) contained in the normalized retention space considered for the calculation of the orthogonality. We propose a Whole Separation Space Scaling (WOSEL) approach that accounts for the whole separation space delineated by the analytical method, and not the sample. This approach enables an orthogonality-based evaluation of the efficiency of the analytical system that is independent of the sample selected. The WOSEL method was compared to two currently used orthogonality approaches through the evaluation of in silico-generated chromatograms and real separations of human biofluids and petroleum samples. WOSEL exhibits sample-to-sample stability values of 3.8% on real samples, compared to 7.0% and 10.1% for the two other methods, respectively. Using real analyses, we also demonstrate that some previously developed approaches can provide misleading conclusions on the overall orthogonality of a two-dimensional chromatographic system.

Metabolite Profiling of the Plasma and Leukocytes of Chronic Myeloid Leukemia Patients.

The discovery of tyrosine kinase inhibitors (TKIs) brought a major breakthrough in the treatment of patients with chronic myeloid leukemia (CML). Pathogenetic CML events are closely linked with the Bcr-Abl protein with tyrosine kinase activity. TKIs block the ATP-binding site; therefore, the signal pathways leading to malignant transformation are no longer active. However, there is limited information about the impact of TKI treatment on the metabolome of CML patients. Using liquid chromatography mass spectrometric metabolite profiling and multivariate statistical methods, we analyzed plasma and leukocyte samples of patients newly diagnosed with CML, patients treated with hydroxyurea and TKIs (imatinib, dasatinib, nilotinib), and healthy controls. The global metabolic profiles clearly distinguished the newly diagnosed CML patients and the patients treated with hydroxyurea from those treated with TKIs and the healthy controls. The major changes were found in glycolysis, the citric acid cycle, and amino acid metabolism. We observed differences in the levels of amino acids and acylcarnitines between those patients responding to imatinib treatment and those who were resistant to it. According to our findings, the metabolic profiling may be potentially used as an additional tool for the assessment of response/resistance to imatinib.

Plasma Acylcarnitines and Amino Acid Levels As an Early Complex Biomarker of Propensity to High-Fat Diet-Induced Obesity in Mice.

Obesity is associated with insulin resistance and impaired glucose tolerance, which represent characteristic features of the metabolic syndrome. Development of obesity is also linked to changes in fatty acid and amino acid metabolism observed in animal models of obesity as well as in humans. The aim of this study was to explore whether plasma metabolome, namely the levels of various acylcarnitines and amino acids, could serve as a biomarker of propensity to obesity and impaired glucose metabolism. Taking advantage of a high phenotypic variation in diet-induced obesity in C57BL/6J mice, 12-week-old male and female mice (n = 155) were fed a high-fat diet (lipids ~32 wt%) for a period of 10 weeks, while body weight gain (BWG) and changes in insulin sensitivity (ΔHOMA-IR) were assessed. Plasma samples were collected before (week 4) and after (week 22) high-fat feeding. Both univariate and multivariate statistical analyses were then used to examine the relationships between plasma metabolome and selected phenotypes including BWG and ΔHOMA-IR. Partial least squares-discrimination analysis was able to distinguish between animals selected either for their low or high BWG (or ΔHOMA-IR) in male but not female mice. Among the metabolites that differentiated male mice with low and high BWG, and which also belonged to the major discriminating metabolites when analyzed in plasma collected before and after high-fat feeding, were amino acids Tyr and Orn, as well as acylcarnitines C16-DC and C18:1-OH. In general, the separation of groups selected for their low or high ΔHOMA-IR was less evident and the outcomes of a corresponding multivariate analysis were much weaker than in case of BWG. Thus, our results document that plasma acylcarnitines and amino acids could serve as a gender-specific complex biomarker of propensity to obesity, however with a limited predictive value in case of the associated impairment of insulin sensitivity.

Combined intervention with pioglitazone and n-3 fatty acids in metformin-treated type 2 diabetic patients: improvement of lipid metabolism.

BACKGROUND: The marine n-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) exert numerous beneficial effects on health, but their potency to improve treatment of type 2 diabetic (T2D) patients remains poorly characterized. We aimed to evaluate the effect of a combination intervention using EPA + DHA and the insulin-sensitizing drug pioglitazone in overweight/obese T2D patients already treated with metformin. METHODS: In a parallel-group, four-arm, randomized trial, 69 patients (66 % men) were assigned to 24-week-intervention using: (i) corn oil (5 g/day; Placebo), (ii) pioglitazone (15 mg/day; Pio), (iii) EPA + DHA concentrate (5 g/day, containing ~2.8 g EPA + DHA; Omega-3), or (iv) pioglitazone and EPA + DHA concentrate (Pio& Omega-3). Data from 60 patients were used for the final evaluation. At baseline and after intervention, various metabolic markers, adiponectin and cytokines were evaluated in serum using standard procedures, EPA + DHA content in serum phospholipids was evaluated using shotgun lipidomics and mass spectrometry, and hyperinsulinemic-euglycemic clamp and meal test were also performed. Indirect calorimetry was conducted after the intervention. Primary endpoints were changes from baseline in insulin sensitivity evaluated using hyperinsulinemic-euglycemic clamp and in serum triacylglycerol concentrations in fasting state. Secondary endpoints included changes in fasting glycemia and glycated hemoglobin (HbA1c), changes in postprandial glucose, free fatty acid and triacylglycerol concentrations, metabolic flexibility assessed by indirect calorimetry, and inflammatory markers. RESULTS: Omega-3 and Pio& Omega-3 increased EPA + DHA content in serum phospholipids. Pio and Pio& Omega-3 increased body weight and adiponectin levels. Both fasting glycemia and HbA1c were increased by Omega-3, but were unchanged by Pio& Omega-3. Insulin sensitivity was not affected by Omega-3, while it was improved by Pio& Omega-3. Fasting triacylglycerol concentrations and inflammatory markers were not significantly affected by any of the interventions. Lipid metabolism in the meal test and metabolic flexibility were additively improved by Pio& Omega-3. CONCLUSION: Besides preventing a modest negative effect of n-3 fatty acids on glycemic control, the combination of pioglitazone and EPA + DHA can be used to improve lipid metabolism in T2D patients on stable metformin therapy. TRIAL REGISTRATION: EudraCT number 2009-011106-42.

Oxidized phosphatidylcholines suggest oxidative stress in patients with medium-chain acyl-CoA dehydrogenase deficiency.

Inborn errors of metabolism encompass a large group of diseases caused by enzyme deficiencies and are therefore amenable to metabolomics investigations. Medium chain acyl-CoA dehydrogenase deficiency (MCADD) is a defect in ß-oxidation of fatty acids, and is one of the most well understood disorders. We report here the use of liquid chromatography-mass spectrometry (LC-MS) based untargeted metabolomics and targeted flow injection analysis-tandem mass spectrometry (FIA-TMS) that lead to discovery of novel compounds of oxidative stress. Dry blood spots of controls (n=25) and patient samples (n=25) were extracted by methanol/water (1/1, v/v) and these supernatants were analyzed by LC-MS method with detection by an Orbitrap Elite MS. Data were processed by XCMS and CAMERA followed by dimension reduction methods. Patients were clearly distinguished from controls in PCA. S-plot derived from OPLS-DA indicated that medium-chain acylcarnitines (octanoyl, decenoyl and decanoyl carnitines) as well as three phosphatidylcholines (PC(16:0,9:0(COOH))), PC(18:0,5:0(COOH)) and PC(16:0,8:0(COOH)) were important metabolites for differentiation between patients and healthy controls. In order to biologically validate these discriminatory molecules as indicators for oxidative stress, a second cohort of individuals were analyzed, including MCADD (n=25) and control (n=250) samples. These were measured by a modified newborn screening method using FIA-TMS (API 4000) in MRM mode. Calculated p-values for PC(16:0,9:0(COOH)), PC(18:0,5:0(COOH)) and PC(16:0,8:0(COOH)) were 1.927×10(-14), 2.391×10(-15) and 3.354×10(-15) respectively. These elevated oxidized phospholipids indeed show an increased presence of oxidative stress in MCADD patients as one of the pathophysiological mechanisms of the disease.