Prostate cancer (PCa) is one of the most common malignancies in men worldwide. Serum prostate specific antigen (PSA) level has been extensively used as a biomarker to detect PCa. However, PSA is not cancer-specific and various non-malignant conditions, including benign prostatic hyperplasia (BPH), can cause a rise in PSA blood levels, thus leading to many false positive results.
Objectives
In this study, we evaluated the potential of urinary metabolomic profiling for discriminating PCa from BPH.
Methods
Urine samples from 64 PCa patients and 51 individuals diagnosed with BPH were analysed using 1H nuclear magnetic resonance (1H-NMR). Comparative analysis of urinary metabolomic profiles was carried out using multivariate and univariate statistical approaches.
Results
The urine metabolomic profile of PCa patients is characterised by increased concentrations of branched-chain amino acids (BCAA), glutamate and pseudouridine, and decreased concentrations of glycine, dimethylglycine, fumarate and 4-imidazole-acetate compared with individuals diagnosed with BPH.
Conclusion
PCa patients have a specific urinary metabolomic profile. The results of our study underscore the clinical potential of metabolomic profiling to uncover metabolic changes that could be useful to discriminate PCa from BPH in a clinical context.
The diagnosis of invasive fungal disease remains challenging in the clinical laboratory. In this paper, the use of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of filamentous fungi as well as its application for antifungal resistance testing and strain typing is evaluated.
Recent Findings
Most studies report very high accuracy for the identification of filamentous fungi by MALDI-TOF MS. Its cost effectiveness, short analysis time, and low error rate and the fact that it can also discriminate between closely related and cryptic species make it appropriate for implementation in the clinical routine. Two drawbacks remain in the availability of extended reference spectra databases and the fact that this technique can only be applied on isolates.
Summary
More work on (simultaneous) antifungal susceptibility testing and strain typing is needed. The application of MALDI-TOF MS directly on clinical specimens would further improve the diagnosis of invasive fungal disease and improve its successful management.
The chemical sensitivity of urine metabolomics analysis is greatly compromised due to the large amounts of inorganic salts in urine (NaCl, KCl), which are detrimental to analytical instrumentation, e.g. chromatographic columns or mass spectrometers. Traditional desalting approaches applied to urine pretreatment suffer from the chemical losses, which reduce the information depth of analysis.
Objectives
We aimed to test a simple approach for the simultaneous preconcentration and desalting of organic solutes in urine based on the collection of induced bursting bubble aerosols above the surface of urine samples.
Method
Bursting bubbles were generated at ambient conditions by feeding gas through an air diffuser at the bottom of diluted (200 times in ultrapure water) urine solution (50–500 mL). Collected aerosols were analyzed by the direct-infusion electrospray ionization mass spectrometry (ESI–MS).
Results
The simultaneous preconcentration (ca. 6–12 fold) and desalting (ca. six–tenfold) of organic solutes in urine was achieved by the bursting bubble sample pretreatment, which allowed ca. three-times higher number of identified urine metabolites by high-resolution MS analysis. No chemical losses due to bubbling were observed. The increased degree of MS data clustering was demonstrated on the principal component analysis of data sets from the urine of healthy people and from the urine people with renal insufficiency. At least ten times higher sensitivity of trace drug detection in urine was demonstrated for clenbuterol and salbutamol.
Conclusion
Our results indicate the high versatility of bubble bursting as a simple pretreatment approach to enhance the chemical depth and sensitivity of urine analysis. The approach could be attractive for personalized medicine as well as for the diagnostics of renal disorders of different etiology (diabetic nephropathy, chronic renal failure, transplant-associated complications, oncological disorders).
Graphical Abstract
Urine desalting and preconcentration in bursting bubbles.
The reports on disseminated candidiasis in dogs so far describe at least one predisposing factor. This case report, however, highlights candidiasis in a dog without any known predisposition.
Patient
A 1.5-year-old intact female Hovawart dog was presented with subcutaneous nodules and polyuria/polydipsia. An excisional biopsy revealed a chronic pyogranulomatous and necrotizing inflammation with mycotic structures. The patient became febrile and lethargic, and developed lameness.
Methods
A physical examination, blood tests, urinalysis, thoracic radiographs, abdominal ultrasonography of the abdomen, fine-needle aspiration biopsies, and a culture of a subcutaneous nodule aspirate were obtained. Selected sections of multiple organs were collected for routine histology postmortem. The isolate and a subcutaneous mass were subjected to molecular identification and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF–MS) analysis.
Results
Clinical, laboratory, and radiological findings were consistent with a granulomatous chronic systemic inflammation. Cytology and histology showed a pyogranulomatous and necrotizing inflammation with myriads of intra- and extra-cellular yeasts and extracellular hyphae. Culture yielded numerous yeast colonies, which appeared Candida albicans–like, but showed a negative serum test and a low identification in API 20 C AUX. Nucleic acid sequences showed homology with the C. albicans-type strain CBS 562. Multilocus sequence typing (MLST) resulted in a new type with designation DST121. The identification of the isolates was confirmed by MALDI-TOF–MS analysis.
Conclusion and Clinical Importance
Future MLST typing and investigation of virulence can provide further evidence whether this MLST-type is associated with clinical cases of disseminated candidiasis without an apparent predisposing condition.
In patients with obstructive jaundice, biliary drainage sometimes fails to result in improvement. A pharmaceutical-grade choleretic herbal medicine, Inchinkoto (ICKT), has been proposed to exert auxiliary effects on biliary drainage; however, its effects are variable among patients.
Objectives
The aim of this study is to explore serum biomarkers that are associated with pharmaceutical efficacy of ICKT.
Methods
Obstructive jaundice patients who underwent external biliary decompression were enrolled (n?=?37). ICKT was given orally 3 times a day at daily dose of 7.5 g. Serum and bile samples were collected before, 3 h after, and 24 h after ICKT administration. The concentrations of total bilirubin, direct bilirubin, and total bile acid in bile specimens were measured. Metabolites in serum samples were comprehensively profiled using LC–MS/MS and GC–MS/MS. Pharmacokinetic analysis of major ICKT components was also performed.
Results
ICKT administration significantly decreased serum ALT and increased bile volume after 24 h. The serum concentrations of ICKT components were not well correlated with the efficacy of ICKT. However, the ratio of 2-hydroxyisobutyric acid to arachidonic acid and the ratio of glutaric acid to niacinamide, exhibited good performance as biomarkers for the efficacy of ICKT on bile flow and ALT, respectively. Additionally, comprehensive correlation analysis revealed that serum glucuronic acid was highly correlated with serum total bilirubin, suggesting that this metabolite may be deeply involved in the pathogenesis of jaundice.
Conclusions
The present study indicates that ICKT is efficacious and provides candidates for predicting ICKT efficacy. Further validation studies are warranted.
Quantification of tetrahydrofolates (THFs), important metabolites in the Wood–Ljungdahl pathway (WLP) of acetogens, is challenging given their sensitivity to oxygen.
Objective
To develop a simple anaerobic protocol to enable reliable THFs quantification from bioreactors.
Methods
Anaerobic cultures were mixed with anaerobic acetonitrile for extraction. Targeted LC–MS/MS was used for quantification.
Results
Tetrahydrofolates can only be quantified if sampled anaerobically. THF levels showed a strong correlation to acetyl-CoA, the end product of the WLP.
Conclusion
Our method is useful for relative quantification of THFs across different growth conditions. Absolute quantification of THFs requires the use of labelled standards.
The purpose of this study is to analyze the current knowledge about matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) identification of clinical yeasts comparing with the ribosomal DNA sequencing identification. We also review the lasts taxonomical and nomenclature changes of clinical yeasts.
Recent Findings
In most studies, the two main commercially available, MALDI Biotyper and VITEK MS, correctly identified more than 90% of yeasts isolates. Most drawbacks are due to low discrimination between closely related species or due to a lack of the reference main spectra (MSP) in the database. The addition of MSPs in the databases improves the performance.
Summary
MALDI-TOF MS has proven to be good for the identification of clinical yeasts and to differentiate some closely related species or species undifferentiated by conventional techniques. However, some species remain difficult to differentiate by this technique and should be identified by ribosomal DNA sequencing. Accurate and rapid yeasts identification could improve the management of patients with yeasts infections.
Aqueous–methanol mixtures have successfully been applied to extract a broad range of metabolites from plant tissue. However, a certain amount of material remains insoluble.
Objectives
To enlarge the metabolic compendium, two ionic liquids were selected to extract the methanol insoluble part of trunk from Betula pendula.
Methods
The extracted compounds were analyzed by LC/MS and GC/MS.
Results
The results show that 1-butyl-3-methylimidazolium acetate (IL-Ac) predominantly resulted in fatty acids, whereas 1-ethyl-3-methylimidazolium tosylate (IL-Tos) mostly yielded phenolic structures. Interestingly, bark yielded more ionic liquid soluble metabolites compared to interior wood.
Conclusion
From this one can conclude that the application of ionic liquids may expand the metabolic snapshot.
Meningitis, a morbidly infectious central nervous system pathology is accompanied by acute inflammation of the meninges, causing raised intracranial pressure linked with serious neurological sequelae.
Objective
To observe the variation in the metabolic profile, that may occur in serum and urine along with CSF in adults using 1H NMR spectroscopy, with an attempt of appropriate and timely treatment regimen.
Methods
The 1H NMR-based metabolomics has been performed in 115 adult subjects for differentiating bacterial meningitis (BM) and tubercular meningitis (TBM).
Results
The discriminant function analysis (DFA) of the three bio-fluids collectively identified 3-hydroxyisovalerate, lactate, glucose, formate, valine, alanine, ketonic bodies, malonate and choline containing compounds (choline and GPC) as significant metabolites among cases versus control group. The differentiation of bacterial meningitis and tuberculous meningitis (BM vs. TBM) can be done on the basis of identification of 3-hydroxyisovalerate, isobutyrate and formate in case of CSF (with a correct classification of 78 %), alanine in serum (correct classification 60 %), valine and acetone in case of urine (correct classification 89.1 %). The NMR spectral bins based orthogonal signal correction principal component analysis score plots of significant metabolites obtained from DFA also provided group classification among cases versus control group in CSF, serum and urine samples. The variable importance in projection scores also identified similar significant metabolites as obtained from DFA, collectively in CSF, serum and urine samples, responsible for differentiation of meningitis.
Conclusion
The CSF contained metabolites which are formed during infection and inflammation, and these were also found in significant quantity in serum and urine samples.
Data processing is one of the biggest problems in metabolomics, given the high number of samples analyzed and the need of multiple software packages for each step of the processing workflow.
Objectives
Merge in the same platform the steps required for metabolomics data processing.
Methods
KniMet is a workflow for the processing of mass spectrometry-metabolomics data based on the KNIME Analytics platform.
Results
The approach includes key steps to follow in metabolomics data processing: feature filtering, missing value imputation, normalization, batch correction and annotation.
Conclusion
KniMet provides the user with a local, modular and customizable workflow for the processing of both GC–MS and LC–MS open profiling data.
A general detrimental effect of smoking during pregnancy on the health of newborn children is well-documented, but the detailed mechanisms remain elusive.
Objectives
Beside the specific influence of environmental tobacco smoke derived toxicants on developmental regulation the impact on the metabolism of newborn children is of particular interest, first as a general marker of foetal development and second due to its potential predictive value for the later occurrence of metabolic diseases.
Methods
Tobacco smoke exposure information from a questionnaire was confirmed by measuring the smoking related metabolites S-Phenyl mercapturic acid, S-Benzyl mercapturic acid and cotinine in maternal urine by LC–MS/MS. The impact of smoking on maternal endogenous serum metabolome and children’s cord blood metabolome was assessed in a targeted analysis of 163 metabolites by an LC–MS/MS based assay. The anti-oxidative status of maternal serum samples was analysed by chemoluminiscence based method.
Results
Here we present for the first time results of a metabolomic assessment of the cordblood of 40 children and their mothers. Several analytes from the group of phosphatidylcholines, namely PCaaC28:1, PCaaC32:3, PCaeC30:1, PCaeC32:2, PCaeC40:1, and sphingomyelin SM C26:0, differed significantly in mothers and children’s sera depending on smoking status. In serum of smoking mothers the antioxidative capacity of water soluble compounds was not significantly changed while there was a significant decrease in the lipid fraction.
Conclusion
Our data give evidence that smoking during pregnancy alters both the maternal and children’s metabolome. Whether the different pattern found in adults compared to newborn children could be related to different disease outcomes should be in the focus of future studies.
Prostate cancer (PCa) is one of the most prevalent cancers in men worldwide. Serum prostate-specific antigen (PSA) remains the most used biomarker in the detection and management of patients with PCa, in spite of the problems related with its low specificity, false positive rate and overdiagnosis. Furthermore, PSA is unable to discriminate indolent from aggressive PCa, which can lead to overtreatment. Early diagnosed and treated PCa can have a good prognosis and is potentially curable. Therefore, the discovery of new biomarkers able to detect clinically significant aggressive PCa is urgently needed.
Methods
This revision was based on an electronic literature search, using Pubmed, with Nuclear Magnetic Resonance (NMR), tissue and prostate cancer as keywords. All metabolomic studies performed in PCa tissues by NMR spectroscopy, from 2007 until March 2018, were included in this review.
Results
In the context of cancer, metabolomics allows the analysis of the entire metabolic profile of cancer cells. Several metabolic alterations occur in cancer cells to sustain their abnormal rates of proliferation. NMR proved to be a suitable methodology for the evaluation of these metabolic alterations in PCa tissues, allowing to unveil alterations in citrate, spermine, choline, choline-related compounds, lactate, alanine and glutamate.
Conclusion
The study of the metabolic alterations associated with PCa progression, accomplished by the analysis of PCa tissue by NMR, offers a promising approach for elucidating biochemical pathways affected by PCa and also for discovering new clinical biomarkers. The main metabolomic alterations associated with PCa development and promising biomarker metabolites for diagnosis of PCa were outlined.
Allograft rejection is still an important complication after kidney transplantation. Currently, monitoring of these patients mostly relies on the measurement of serum creatinine and clinical evaluation. The gold standard for diagnosing allograft rejection, i.e. performing a renal biopsy is invasive and expensive. So far no adequate biomarkers are available for routine use.
Objectives
We aimed to develop a urine metabolite constellation that is characteristic for acute renal allograft rejection.
Methods
NMR-Spectroscopy was applied to a training cohort of transplant recipients with and without acute rejection.
Results
We obtained a metabolite constellation of four metabolites that shows promising performance to detect renal allograft rejection in the cohorts used (AUC of 0.72 and 0.74, respectively).
Conclusion
A metabolite constellation was defined with the potential for further development of an in-vitro diagnostic test that can support physicians in their clinical assessment of a kidney transplant patient.
Mass spectrometry is the current technique of choice in studying drug metabolism. High-resolution mass spectrometry in combination with MS/MS gas-phase experiments has the potential to contribute to rapid advances in this field. However, the data emerging from such fragmentation spectral files pose challenges to downstream analysis, given their complexity and size.
Objectives
This study aims to detect and visualize antihypertensive drug metabolites in untargeted metabolomics experiments based on the spectral similarity of their fragmentation spectra. Furthermore, spectral clusters of endogenous metabolites were also examined.
Methods
Here we apply a molecular networking approach to seek drugs and their metabolites, in fragmentation spectra from urine derived from a cohort of 26 patients on antihypertensive therapy. The mass spectrometry data was collected on a Thermo Q-Exactive coupled to pHILIC chromatography using data dependent analysis (DDA) MS/MS gas-phase experiments.
Results
In total, 165 separate drug metabolites were found and structurally annotated (17 by spectral matching and 122 by classification based on a clustered fragmentation pattern). The clusters could be traced to 13 drugs including the known antihypertensives verapamil, losartan and amlodipine. The molecular networking approach also generated clusters of endogenous metabolites, including carnitine derivatives, and conjugates containing glutamine, glutamate and trigonelline.
Conclusions
The approach offers unprecedented capability in the untargeted identification of drugs and their metabolites at the population level and has great potential to contribute to understanding stratified responses to drugs where differences in drug metabolism may determine treatment outcome.
Amino acid analysis in biological fluids is essential for the study of inborn errors of metabolism (IEM) and other diseases.
Objectives
Our aim was to develop a UPLC-MS/MS procedure for the analysis of 25 amino acids and identification of 17 related compounds.
Methods
Sample treatment conditions were optimized for plasma, urine, cerebrospinal fluid (CSF) and dried blood spots. Amino acids and related compounds were analyzed on a Waters ACQUITY UPLC H-class instrument with a reversed-phase C-18 column using water and acetonitrile with 0.1% formic acid as the mobile phases (run time?=?9 min). The detection was performed with a Waters Xevo TQD triple-quadrupole mass spectrometer using positive electrospray ionization in the multiple reaction monitoring mode.
Results
The method linearity, intra-assay and inter-assay precision, detection limit, quantification limit and trueness analysis displayed adequate results in both physiological and pathological conditions. Method comparison was performed between UPLC-MS/MS and ion exchange chromatography (IEC) with ninhydrin derivatization, and the methods showed good agreement, except for 4-hydroxyproline, aspartate and citrulline. Paediatrics age-related reference values in plasma, urine and CSF were established and patients with different IEM were easily identified.
Conclusion
We report a modified UPLC-MS/MS procedure for the analysis of 42 amino acids and related compounds in different specimens. The method is fast, sensitive and robust, and it has been validated to be an alternative to the traditional IEC procedure as the routine method used in metabolic laboratories. The method greatly decreases the run time of the analysis while displaying good metrological results.
Ketosis is a common metabolic disorder, which is characterized by elevated concentrations of ketone bodies or ketoacids in three body fluids including blood, urine, and milk. Two of the ketones including β-hydroxybutyric acid and acetoacetic acid are strong acids which at high concentrations trigger ketoacidosis influencing physiological functions of various tissues and organs.
Objectives
The objectives of this study were to: (1) investigate mineral alterations in both serum and urine of preketotic, ketotic, and postketotic cows, (2) identify potential predictive and diagnostic mineral biomarkers for ketosis in serum and urine, and (3) better understand the role of minerals in the pathobiology of the disease.
Methods
Inductively coupled plasma mass spectrometry metallotyping was performed in the serum and urine of six cases of ketosis and 20 healthy controls cows at ?8 and ?4 weeks prepartum, at disease diagnosis week, and at +4 and +8 weeks postpartum.
Results
Data showed that concentrations of aluminum (Al), iron (Fe), manganese (Mn), and arsenic (As) were greater (P?<?0.001) in the serum of preketotic, ketotic and postketotic cows at most of the tested time points. Moreover, boron (B) and Al as well as calcium (Ca), phosphorus (P), potassium (K), and magnesium (Mg) were found to be elevated in the urine of preketotic and postketotic cows (P?<?0.001).
Conclusions
It is concluded that alterations of mineral elements observed in the serum and urine of preketotic, ketotic, and postketotic cows might be related to the state of chronic acidosis in those cows. The mineral elements identified in both serum and urine can be used as biomarkers to early diagnose ketosis at its pre-subclinical state and develop preventive interventions in the future.
Sporotrichosis, the disease caused by Sporothrix spp, ranges from subcutaneous infections to the severe disseminated or invasive diseases. The taxonomy of Sporothrix has been revised. The subcutaneous disease is suspected easily, but the extra-cutaneous disease is diagnosed by chance or with high suspicion. This review provides the overview of currently available diagnostic techniques.
Recent Finding
Enzyme-linked immunosorbent assay (ELISA) or latex agglutination test with partially purified antigens helps in the diagnosis of extra-cutaneous sporotrichosis. Molecular methods have been used for the identification and typing of the fungus. Calmodulin, beta tubulin, translation elongation factor and chitin synthase genes are targeted for species differentiation. MALDI-TOF MS has been standardized to identify the species.
Summary
PCR-based molecular techniques and matrix-assisted laser desorption ionisation time of flight mass spectroscopy (MALDI-TOF MS) help in the identification of Sporothrix species, whereas ELISA helps in diagnosing extra-cutaneous form. Utility of molecular techniques for detection of Sporothrix directly from clinical specimen needs to be evaluated.
Tandem mass spectrometry (MS/MS) has been widely used for identifying metabolites in many areas. However, computationally identifying metabolites from MS/MS data is challenging due to the unknown of fragmentation rules, which determine the precedence of chemical bond dissociation. Although this problem has been tackled by different ways, the lack of computational tools to flexibly represent adjacent structures of chemical bonds is still a long-term bottleneck for studying fragmentation rules.
Objectives
This study aimed to develop computational methods for investigating fragmentation rules by analyzing annotated MS/MS data.
Methods
We implemented a computational platform, MIDAS-G, for investigating fragmentation rules. MIDAS-G processes a metabolite as a simple graph and uses graph grammars to recognize specific chemical bonds and their adjacent structures. We can apply MIDAS-G to investigate fragmentation rules by adjusting bond weights in the scoring model of the metabolite identification tool and comparing metabolite identification performances.
Results
We used MIDAS-G to investigate four bond types on real annotated MS/MS data in experiments. The experimental results matched data collected from wet labs and literature. The effectiveness of MIDAS-G was confirmed.
Conclusion
We developed a computational platform for investigating fragmentation rules of tandem mass spectrometry. This platform is freely available for download.
