Wide metabolite coverage LC-MS/MS assay for the diagnosis of inherited metabolic disorders in urine.

OBJECTIVE: The laboratory diagnosis of inherited metabolic disorders (IMD) has undergone significant development in recent decades, mainly due to the use of mass spectrometry, which allows rapid multicomponent analysis of a wide range of metabolites. Combined with advanced software tools, the diagnosis becomes more efficient as a benefit for both physicians and patients. METHODS: A hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry assay for determination of urinary purines, pyrimidines, N-acylglycines, N-acetylated amino acids, sugars, sugar alcohols and other diagnostically important biomarkers was developed and validated. Evaluation of the results consisting of utilisation of robust scaling and advanced visualization tools is simple and even suitable for urgent requirements. RESULTS: The developed method, covering 65 biomarkers, provides a comprehensive diagnostic platform for 51 IMD. For most analytes, linearity with R2 > 0.99, intra and inter-day accuracy between 80 and 120 % and precision lower than 20 % were achieved. Diagnostic workflow was evaluated on 47 patients and External Quality Assurance samples involving a total of 24 different IMD. Over seven years, more than 2300 urine samples from patients suspected for IMD have been routinely analysed. CONCLUSIONS: This method offers the advantage of a broad coverage of intermediate metabolites of interest and therefore may be a potential alternative and simplification for clinical laboratories that use multiple methods for screening these markers.

The dementia-friendly community and its implications for healthcare.

Principal goal of the article is to inform health care professionals about the concept of dementia-friendly community (DFC) reflecting the contemporary thinking of living with dementia, i.e. dementia as a disability, equal human rights, a sense of meaning. Experiences from abroad and from the Czechia are discussed with special attention to implementation of this concept in the health care facilities. The National Action Plan for Alzheimer´s Disease 2020-2030 (NAPAN) is presented, which shows that the Czech Government, ministries, specialists, care providers, informal careers and even the people with dementia worked together on the improvement in this field. The purpose of this paper is to bring the principles of DFC closer to the physicians and other health care professionals and support friendly and helpful approach to people living with dementia when providing health care to them. The multidisciplinary cooperation of GPs, geriatricians, neurologists, psychiatrists etc. is emphasized. An example of a regional project in Middle Bohemia is presented looking for criteria and ways of evaluation of the effect of DFC.

SLIDE-Novel Approach to Apocrine Sweat Sampling for Lipid Profiling in Healthy Individuals.

We designed a concept of 3D-printed attachment with porous glass filter disks-SLIDE (Sweat sampLIng DevicE) for easy sampling of apocrine sweat. By applying advanced mass spectrometry coupled with the liquid chromatography technique, the complex lipid profiles were measured to evaluate the reproducibility and robustness of this novel approach. Moreover, our in-depth statistical evaluation of the data provided an insight into the potential use of apocrine sweat as a novel and diagnostically relevant biofluid for clinical analyses. Data transformation using probabilistic quotient normalization (PQN) significantly improved the analytical characteristics and overcame the 'sample dilution issue' of the sampling. The lipidomic content of apocrine sweat from healthy subjects was described in terms of identification and quantitation. A total of 240 lipids across 15 classes were identified. The lipid concentrations varied from 10-10 to 10-4 mol/L. The most numerous class of lipids were ceramides (n = 61), while the free fatty acids were the most abundant ones (average concentrations of 10-5 mol/L). The main advantages of apocrine sweat microsampling include: (a) the non-invasiveness of the procedure and (b) the unique feature of apocrine sweat, reflecting metabolome and lipidome of the intracellular space and plasmatic membranes. The SLIDE application as a sampling technique of apocrine sweat brings a promising alternative, including various possibilities in modern clinical practice.

Directly Measured Adherence to Treatment in Chronic Heart Failure: LEVEL-CHF Registry.

BACKGROUND: Currently, most available data on the medication adherence of patients with chronic heart failure are based on indirect methods. We examined the level of adherence to medical therapy using a direct method - serum drug level testing. METHODS: We carried out a prospective single-centre registry of patients with chronic heart failure (LEVEL-CHF registry), in whom we analysed serum levels of the medications prescribed for the treatment of heart failure: angiotensin converting enzyme inhibitors, angiotensin receptor blockers, beta-blockers and mineralocorticoid receptor antagonists. We labelled a patient as non-adherent if at least one serum level of a prescribed drug was unmeasurable (below the detection limit). Patients with all tested drugs identifiable in serum were labelled as adherent. We enrolled 274 patients (208 men and 66 women) mean age 62 years. RESULTS: 82.5% of patients were adherent and 17.5% non-adherent to prescribed medications. 3.6% were completely non-adherent without any detectable drugs in serum. Patients aged <60 years were more likely to be non-adherent than older patients (OR 2.15). No other clinical or laboratory parameters predicted non-adherence. CONCLUSIONS: A significant proportion of outpatients with chronic heart failure were non-adherent to treatment when assessed by a direct method of serum drug level testing. Non-adherence was more likely in younger patients.

A newborn screening approach to diagnose 3-hydroxy-3-methylglutaryl-CoA lyase deficiency.

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.

Workplace stress and employees' well-being: Evidence from long-term care in the Czech Republic.

OBJECTIVES: The aim of this study was to verify the Effort-Reward Imbalance (ERI) model, which served as the basic concept of mapping workplace stress, on the sample of Czech professional caregivers. ERI model examines the relationship between the long-term subjectively perceived level of workers' effort and rewards and analyses the physical and psychosocial consequences of the (im)balance. METHODS: The verification of ERI model in combination with well-being (and its psychosocial consequences) was conducted on a sample of Czech professionals caring for older people in 2014 (N = 265). The survey included 12 facilities providing health and social care services for older people. Facilities were chosen through purposive sampling and snowball technique. The sample was divided into the following subgroups: professionals working in residential or field services and medical or social workers. RESULTS: Results showed that the majority (57%) of professional caregivers in both residential and field services suffered from imbalance caused by higher effort and lower rewards. Subgroup of medical workers in long-term care institutions formed the most demanded group with the highest imbalance between work effort and rewards (68%). This discrepancy was reflected in a reduction of their well-being. This effect was most evident by the medical workers in home care. Well-being within this group was more than five times lower compared to other groups with ERI imbalance. Also, a group of social workers in institutions came out as a high-risk group in this regard. DISCUSSION: The level of imbalance differed among the defined groups. The data obtained verified the known facts about the adverse work situation of professionals in long-term care in the contemporary Czech environment. The outputs correspond to foreign studies and confirm the validity of ERI model on the sample of Czech professionals in the long-term care.

Newborn foal with atypical myopathy.

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.

Radio-sensitizing effects of VE-821 and beyond: Distinct phosphoproteomic and metabolomic changes after ATR inhibition in irradiated MOLT-4 cells.

Current anti-cancer strategy takes advantage of tumour specific abnormalities in DNA damage response to radio- or chemo-therapy. Inhibition of the ATR/Chk1 pathway has been shown to be synthetically lethal in cells with high levels of oncogene-induced replication stress and in p53- or ATM- deficient cells. In the presented study, we aimed to elucidate molecular mechanisms underlying radiosensitization of T-lymphocyte leukemic MOLT-4 cells by VE-821, a higly potent and specific inhibitor of ATR. We combined multiple approaches: cell biology techniques to reveal the inhibitor-induced phenotypes, and quantitative proteomics, phosphoproteomics, and metabolomics to comprehensively describe drug-induced changes in irradiated cells. VE-821 radiosensitized MOLT-4 cells, and furthermore 10 µM VE-821 significantly affected proliferation of sham-irradiated MOLT-4 cells. We detected 623 differentially regulated phosphorylation sites. We revealed changes not only in DDR-related pathways and kinases, but also in pathways and kinases involved in maintaining cellular metabolism. Notably, we found downregulation of mTOR, the main regulator of cellular metabolism, which was most likely caused by an off-target effect of the inhibitor, and we propose that mTOR inhibition could be one of the factors contributing to the phenotype observed after treating MOLT-4 cells with 10 µM VE-821. In the metabolomic analysis, 206 intermediary metabolites were detected. The data indicated that VE-821 potentiated metabolic disruption induced by irradiation and affected the response to irradiation-induced oxidative stress. Upon irradiation, recovery of damaged deoxynucleotides might be affected by VE-821, hampering DNA repair by their deficiency. Taken together, this is the first study describing a complex scenario of cellular events that might be ATR-dependent or triggered by ATR inhibition in irradiated MOLT-4 cells. Data are available via ProteomeXchange with identifier PXD008925.

Hemodiafiltration and plasma levels of axitinib in a patient with metastatic renal clear cell carcinoma.

BACKGROUND: The standard treatment for metastatic renal cancer is based on vascular endothelial growth factor (VEGF) and mammalian target of rapamycin (mTor) inhibitors. Compared to other advanced tumors, the treatment of renal cancer is highly affected by impaired renal function; therefore, patients with severe renal insufficiency, including patients on hemodialysis, are generally excluded from clinical trials. CASE REPORT: In the present manuscript we present the case of a renal cancer patient who underwent bilateral nephrectomy and received two lines of treatment. We hypothesized that axitinib, a tyrosine kinase inhibitor, would have a similar plasma concentration to patients without hemodialysis and that the levels before and after hemodiafiltration will not differ significantly, as observed in other targeted therapies. CONCLUSION: The observed axitinib concentrations were at least an order of magnitude lower than expected based on the literature and measurements in other patients. The present case report indicates a potential risk of axitinib underdosing in patients on hemodiafiltration with the standard dose; therefore, drug dosage may need to be corrected based on the plasma levels of axitinib.

Ultrafast Online SPE-MS/MS Method for Quantification of 3 Tyrosine Kinase Inhibitors in Human Plasma.

BACKGROUND: With an increasing number of cancer patients receiving tyrosine kinase inhibitors (TKIs), therapeutic drug monitoring of these molecules is becoming more widespread today. It is mainly based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) methods with typical run times of several minutes. In an online solid phase extraction-MS/MS (SPE-MS/MS) system, the chromatography column is replaced with a reusable solid phase extraction (SPE) cartridge and the analysis time is shortened to less than half a minute. The aim of this study was to develop such a method and test the performance of this high-throughput system in the analysis of imatinib (IMA), nilotinib (NIL), and lapatinib (LAP) in human plasma. METHODS: Samples were prepared by simple protein precipitation with methanol containing deuterated internal standards. After centrifugation, the supernatant was diluted 10 fold with a mixture of methanol and water (1:1). A C4 cartridge was used for SPE and the analytes were eluted by acetonitrile. All the analytes were measured within a wide calibration range (50-5000 ng/mL for nilotinib and imatinib, 100-10,000 ng/mL for lapatinib). The method was compared with the LC-MS/MS method by the analysis of 176 clinical samples. RESULTS: Intraday and interday inaccuracies within 15% and a coefficient of variation less than 15% were achieved for all the TKIs that were measured. Even though the matrix effects were higher in comparison with LC-MS/MS methods, their effect on the performance of the method was eliminated by the usage of deuterated internal standards. The total run time of the new method was 29 seconds for one analysis and the results were fully comparable with LC-MS/MS. CONCLUSIONS: Routine clinical practice requiring high-throughput methods for therapeutic drug monitoring of TKIs may benefit from the online SPE-MS/MS method that provides fast, low-cost analysis, and results that are comparable with conventional methods.

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.

Family Carers' Perspectives on Integrated Community Care in the Czech Republic.

AIM: The problem of family care for people dependent on another person has only recently become a focus of research. As demand for health and social services has not been adequately met by public service providers, growing attention has been given to informal care and the integration of families within systems of health and social care at the community level. This paper presents the results of a survey on informal carers' views and opinions under the current conditions of social support in the Czech Republic. The survey was based on theoretical concepts of caring societies, deinstitutionalization, refamilization, and integrated community care, and aimed to shed light on caring families' experiences and needs in the Czech Republic. METHODS: Family lay carers completed an original self-administered questionnaire. A convenient sample of 200 family carers was selected. RESULTS: The survey collected information about the most influential factors in determining whether the families continue to provide care for their relatives in the household. More than 50% of the caregivers provide care from moral and emotional reasons. Financial problems, risk of losing their jobs, and further deterioration of health of the person they care for emerged as key risk factors, but overall, determination among carers to continue providing care "at any cost" was high (53%). CONCLUSIONS: Involving local communities and services, e.g. general practitioners (GPs) to a greater extent in the coordination of various social and health services, and in support mechanisms at the juncture between informal and formal care would make it easier for family carers to continue providing long-term care.

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.

Untargeted metabolomic analysis of urine samples in the diagnosis of some inherited metabolic disorders.

BACKGROUND: Metabolomics is becoming an important tool in clinical research and the diagnosis of human diseases. It has been used in the diagnosis of inherited metabolic disorders with pronounced biochemical abnormalities. The aim of this study was to determine if it could be applied in the diagnosis of inherited metabolic disorders (IMDs) with less clear biochemical profiles from urine samples using an untargeted metabolomic approach. METHODS: A total of 14 control urine samples and 21 samples from infants with cystinuria, maple syrup urine disease, adenylosuccinate lyase deficiency and galactosemia were tested. Samples were analyzed by liquid chromatography on aminopropyl column in aqueous normal phase separation system using gradient elution of acetonitrile/ammonium acetate. Detection was performed by time-of-flight mass spectrometer fitted with electrospray ionisation in positive mode. The data were statistically processed using principal component analysis (PCA), principal component discriminant function analysis (PCA-DFA) and partial least squares (PLS) regression. RESULTS: All patient samples were first distinguished from controls using unsupervised PCA. Discrimination of the patient samples was then unambiguously verified using supervised PCA-DFA. Known markers of the diseases in question were successfully confirmed and a potential new marker emerged from the PLS regression. CONCLUSION: This study showed that untargeted metabolomics can be applied in the diagnosis of mild IMDs with less clear biochemical profiles.

The attitudes of older adults living in institutions and their caregivers to ageing.

OBJECTIVES: The aim of this study was to explore the attitudes of older people living in institutions and their caregivers to ageing. Recent outcomes showed prevailing negative social stereotype to ageing in CR. METHODS: The Attitudes to Ageing Questionnaire (AAQ-24) was used in two waves of data collection to measure attitudes of 400 randomly selected residents of 19 Senior Residential Homes. The reduced sample of 220 seniors and 276 professional carers employed at twelve Senior Residential Homes completed 12 items of general form (AAQ-12). All respondents expressed their agreement or disagreement with the statements presented in the questionnaire regarding positive or negative attitudes to ageing. RESULTS: The AAQ total score proved significant influence of gender, having children, self-perceived health, depression, and quality of life. Subscale scores (psychosocial losses, physical changes, psychological growth) were significantly influenced by gender, age, activities limitations, having own children, depression, self-perceived health status, and quality of life. Globally, the attitudes of professional caregivers to ageing were more positive compared to the attitudes of older people living in institutions. Older adults showed higher agreement with negative statements about ageing. There was no difference between professional caregivers and older people in the positive attitudes to ageing expressed as the growth potential. Physical activity, wisdom, better ability to cope with life and contacting young generation were effective in the positive attitudes of both groups.

Targeted metabolomic analysis of plasma samples for the diagnosis of inherited metabolic disorders.

Metabolomics has become an important tool in clinical research and diagnosis of human diseases. In this work we focused on the diagnosis of inherited metabolic disorders (IMDs) in plasma samples using a targeted metabolomic approach. The plasma samples were analyzed with the flow injection analysis method. All the experiments were performed on a QTRAP 5500 tandem mass spectrometer (AB SCIEX, U.S.A.) with electrospray ionization. The compounds were measured in a multiple reaction monitoring mode. We analyzed 50 control samples and 34 samples with defects in amino acid metabolism (phenylketonuria, maple syrup urine disease, tyrosinemia I, argininemia, homocystinuria, carbamoyl phosphate synthetase deficiency, ornithine transcarbamylase deficiency, nonketotic hyperglycinemia), organic acidurias (methylmalonic aciduria, propionic aciduria, glutaric aciduria I, 3-hydroxy-3-methylglutaric aciduria, isovaleric aciduria), and mitochondrial defects (medium-chain acyl-coenzyme A dehydrogenase deficiency, carnitine palmitoyltransferase II deficiency). The controls were distinguished from the patient samples by principal component analysis and hierarchical clustering. Approximately 80% of patients were clearly detected by absolute metabolite concentrations, the sum of variance for first two principle components was in the range of 44-55%. Other patient samples were assigned due to the characteristic ratio of metabolites (the sum of variance for first two principle components 77 and 83%). This study has revealed that targeted metabolomic tools with automated and unsupervised processing can be applied for the diagnosis of various IMDs.

Proving tumour cells by acute nutritional/energy deprivation as a survival threat: a task for microscopy.

Malignant cells appear to possess a special aptitude for survival. We attempted to prove this in vitro by acute nutritional and energy deprivation as a survival threat. A phosphate-buffered saline (PBS) survival test in cell culture allowed static observations. These were supplemented by classic and quantitative phase-contrast time-lapse microscopy. From one normal and four neoplastic cell populations, no cells survived 77 hours of exposure to PBS. Only G3S2 derived from a human breast carcinoma survived 60 hours. Cells in sparse culture were more vulnerable than those in dense. Epithelial cells were more vigorous than mesenchymal cells. Cells of greater malignancy resisted longer. Evaluation in culture as detailed by digital holographic microscopy (DHM) revealed an increase in the compactness of the intracellular mass motility from normal to metastasizing mesenchymal cells, thus reaching the level of epithelial G3S2 cells. Studying the PBS survival test with DHM opens a new approach to investigations of the structural integrity of neoplastic cells.

Functional status and depressive symptoms among older adults from residential care facilities in the Czech Republic.

BACKGROUND: Depressive symptoms are common among older adults, particularly those living in long-term care facilities. However, little is known about factors associated with depressive symptoms among long-term care residents in the Czech Republic and in other Eastern European countries. Moreover, the role of mobility and pain in depressive symptoms among long-term care residents is relatively understudied. OBJECTIVE: We examined the relationship between functional status and depressive symptoms in 308 older adults from residential care facilities (RCFs) in the Czech Republic. METHOD: We used baseline data from two randomized controlled trials testing the effects of dance and reminiscence therapies on quality of life in older RCF residents. Functional status was measured as cognitive function, general ability to perform basic Activities of Daily Living (ADLs), mobility, and functional limitation by pain. Depressive symptoms were measured using the 15-item Geriatric Depression Scale. RESULTS: In multiple regression analyses adjusted for sociodemographic factors and taking antidepressants, we found that cognitive function and functional limitation by pain were most strongly associated with depressive symptoms. The ability to perform basic ADLs and mobility were also related to depressive symptoms. CONCLUSION: Our findings suggest factors that may be important in efforts to improve psychological well-being in this population.