Expression and 7-day time course of circulating microRNAs in septic patients treated with nephrotoxic antibiotic agents.

BACKGROUND: Through regulation of signaling pathways, microRNAs (miRNAs) can be involved in sepsis and associated organ dysfunction. The aims of this study were to track the 7-day time course of blood miRNAs in patients with sepsis treated with vancomycin, gentamicin, or a non-nephrotoxic antibiotic and miRNA associations with neutrophil gelatinase-associated lipokalin (NGAL), creatinine, procalcitonin, interleukin-6, and acute kidney injury (AKI) stage. METHODS: Of 46 adult patients, 7 were on vancomycin, 20 on gentamicin, and 19 on another antibiotic. Blood samples were collected on days 1, 4, and 7 of treatment, and miRNAs were identified using quantitative reverse transcription PCR. RESULTS: The results showed no relationship between miRNA levels and biochemical variables on day 1. By day 7 of gentamicin treatment miR-15a-5p provided good discrimination between AKI and non-AKI (area under curve, 0.828). In patients taking vancomycin, miR-155-5p and miR-192-5p positively correlated with creatinine and NGAL values, and miR-192-5p and miR-423-5p positively correlated with procalcitonin and interleukin-6 in patients treated with a non-nephrotoxic antibiotic. In patients together we found positive correlation between miR-155-5p and miR-423-5p and all biochemical markers. CONCLUSION: The results suggest that these four miRNAs may serve as diagnostic or therapeutic tool in sepsis, renal injury and nephrotoxic treatment. TRIAL REGISTRATION: ClinicalTrials.gov , ID: NCT04991376 . Registered on 27 July 2021.

Acute Kidney Injury in Septic Patients Treated by Selected Nephrotoxic Antibiotic Agents-Pathophysiology and Biomarkers-A Review.

Acute kidney injury is a common complication in critically ill patients with sepsis and/or septic shock. Further, some essential antimicrobial treatment drugs are themselves nephrotoxic. For this reason, timely diagnosis and adequate therapeutic management are paramount. Of potential acute kidney injury (AKI) biomarkers, non-protein-coding RNAs are a subject of ongoing research. This review covers the pathophysiology of vancomycin and gentamicin nephrotoxicity in particular, septic AKI and the microRNAs involved in the pathophysiology of both syndromes. PubMED, UptoDate, MEDLINE and Cochrane databases were searched, using the terms: biomarkers, acute kidney injury, antibiotic nephrotoxicity, sepsis, miRNA and nephrotoxicity. A comprehensive review describing pathophysiology and potential biomarkers of septic and toxic acute kidney injury in septic patients was conducted. In addition, five miRNAs: miR-15a-5p, miR-192-5p, miR-155-5p, miR-486-5p and miR-423-5p specific to septic and toxic acute kidney injury in septic patients, treated by nephrotoxic antibiotic agents (vancomycin and gentamicin) were identified. However, while these are at the stage of clinical testing, preclinical and clinical trials are needed before they can be considered useful biomarkers or therapeutic targets of AKI in the context of antibiotic nephrotoxicity or septic injury.

Reference intervals of plasma matrix metalloproteinases 2, 3, and 9 and serum asymmetric dimethylarginine levels.

OBJECTIVE: The present study aimed to verify the reference intervals of plasma matrix metalloproteinases (MMPs) 2, 3, and 9 and serum asymmetric dimethylarginine (ADMA) in a healthy population with an average age corresponding to that of patients with cardiovascular diseases. METHODS: The study included 180 healthy volunteers. Plasma MMP-2, MMP-3, MMP-9, and serum ADMA levels were determined using an enzyme-linked immunosorbent assay. These levels were analyzed for association with age and gender. The Cbstat5, R software, and NCSS 2007 programs were used for statistical analysis. RESULTS: The average volunteer age was 47.4 years in the group in which MMP-3 and ADMA were analyzed, 40.3 years in the MMP-9 group, and 47.8 years for the MMP-2 group. Serum ADMA levels were determined to be independent of age and gender. Plasma MMP-2 levels were significantly correlated with age (p = 0.001), with lower levels detected in persons ≤ 49 years of age. Plasma MMP-3 was significantly associated with both age (p < 0.0001) and gender, with lower levels detected in persons of ≤ 47 years of age and among women. Plasma MMP-9 levels were not age dependent, but were associated with gender (p = 0.014), showing lower levels in women. CONCLUSIONS: Reference intervals of heparin-plasma MMP-2, MMP-3, and MMP-9 and serum ADMA levels were determined. MMP-2 and MMP-3 levels were found to be age dependent, and MMP-3 and MMP-9 levels were gender dependent.

Lacrimal sac dacryoliths (86 samples): chemical and mineralogic analyses.

BACKGROUND: Because dacryoliths occur at low frequency, few studies have focused on their composition. We aimed to present findings from morphological, chemical, and mineralogic analysis of 86 dacryoliths. METHODS: We studied 86 dacryoliths obtained during 832 dacryocystorhinostomies (DCR) performed for postsaccal obstruction. We examined the samples with atomic infrared spectrometry (80 samples), amino acid analysis (17 samples), scanning electron microscopy, and an electron microprobe with an energy dispersive detector (seven samples). RESULTS: Dacryoliths were found in 86/832 DCRs (10.3 %), mostly in patients with primary acquired nasolacrimal duct obstruction. All the dacryoliths were soft, composed of organic material, including proteins and mucoproteins, with approximately 20 % amino acid content. There were no "hard" dacryoliths composed of calcium phosphate. The stones were composed of lobes and lobules built on an amorphous core material with small cavities, probably as a result of various chemical processes that produced a gaseous product. The most frequent elements found in inorganic inclusions were silicon, magnesium, sulfur, potassium, calcium, sodium, and chlorine. Also, some particles had high contents of bismuth, titanium, iron, and organic fibers. The fibers found in the core of dacryoliths suggested a potential origin from cotton swabs used in cosmetics. CONCLUSION: Dacryoliths are composed almost exclusively of organic material, including proteins and mucoproteins, with approximately 20 % amino acid content.

Corrigendum: Secretoneurin levels are higher in dilated cardiomyopathy than in ischaemic cardiomyopathy: preliminary results.

[This corrects the article DOI: 10.3389/fcvm.2023.1297900.].

Prevention of sarcopenia in patients with obesity after bariatric and metabolic surgery: The effect of programmed training on the muscle tissue and anthropometric functions - A randomized controlled trial (SarxOb study protocol).

Obesity is a serious metabolic disease that significantly increases cardiovascular risks and other health complications. Sarcopenia is an independent risk factor for morbidity and mortality in patients suffering from obesity that increases the health risks and is associated with cardiac, respiratory and other diseases.  Bariatric and metabolic surgery (BMS) leads to significant changes in body composition. Our pilot study showed that bariatric patients are at risk of sarcopenia after BMS. This finding resulted in a hypothesis that an exercise plan in the experimental group will lead to postural stabilization and a lower decline in muscle homotopy, further leading to a greater reduction in fat mass and a positive effect of exercise on skeletal muscle volume and strength and endocrine-metabolic function. The aim of the present study is to determine the effect of programmed aerobic and strength training on muscle function, volume, and morphology in patients after BMS. The study is a single-center, randomized clinical trial after sleeve gastrectomy focused on muscle tissue. The experimental group will perform targeted physical activity once a week for 12 months and the training plan will include anaerobic and aerobic components. Magnetic resonance imaging of skeletal muscles will be correlated with the values of densitometry examination and changes in body composition, certain blood parameters of myokines, biomechanical analysis of movement abnormalities, and behavioral and dietary counseling. This study will address the research questions about the effect of programmed training on muscle tissue and muscular functions after BMS.

Secretoneurin levels are higher in dilated cardiomyopathy than in ischaemic cardiomyopathy: preliminary results.

Background: Secretoneurin (SN) is a neuropeptide with potential utility as a biomarker of cardiovascular episodes. The main effect of SN is mediated through its inhibition of calmodulin-dependent kinase II (CaMKII), which influences calcium handling. We aimed to associate the levels of SN in plasma with different causes of heart failure. Methods: We prospectively enrolled consecutive patients with ischaemic (ICM) and dilated (DCM) cardiomyopathy from the outpatient heart failure clinic and healthy individuals. SN was analysed from venous blood by use of the ELISA method. SN plasma levels were compared in DCM, ICM and healthy individuals with non-parametric tests. Results: A total of 53 patients (81.1% male, 18.9% female; mean age 67.9 ± 12.6 years) and 34 healthy individuals (38% male, 62% female) were included in the analysis. Plasma SN levels were significantly higher in the dilated cardiomyopathy (38.8 ± 27 pmol/L) as compared with the ischaemic cardiomyopathy (19.7 ± 22.6 pmol/L) group (P = 0.006). There was no significant difference between females vs. males (27.1 ± 23 vs. 25.5 ± 26.2 pmol/L, P = NS). Plasma SN levels allowed DCM and ICM to be differentiated with 88% sensitivity and 61% specificity (P = 0.007), the cut of value is 13.3 pmol/L. Plasma SN levels differed significantly between healthy volunteers and both ICM (P < 0.0001) and DCM (P = 0.049). Plasma SN levels did not differ according to age and were not associated with comorbidities, left ventricular ejection fraction, heart failure medication, troponin, creatinine, or natriuretic peptide plasma levels. Conclusion: Plasma secretoneurin levels differed significantly in DCM vs. ICM, being higher in the former. Based on plasma SN levels, discrimination between DCM and ICM might be possible. Healthy individuals produce higher SN plasma levels than stable HFrEF patients.

Study of disulfide reduction and alkyl chloroformate derivatization of plasma sulfur amino acids using gas chromatography-mass spectrometry.

Four disulfide-reducing agents, dithiothreitol (DTT), 2,3-dimercaptopropanesulfonate (DMPS), and the newly tested 2-mercaptoethanesulfonate (MESNA) and Tris(hydroxypropyl)phosphine (THP), were investigated in detail for release of sulfur amino acids in human plasma. After protein precipitation with trichloroacetic acid (TCA), the plasma supernatant was treated with methyl, ethyl, or propyl chloroformate via the well-proven derivatization-extraction technique and the products were subjected to gas chromatographic-mass spectrometric (GC-MS) analysis. All the tested agents proved to be rapid and effective reducing agents for the assay of plasma thiols. When compared with DTT, the novel reducing agents DMPS, MESNA, and THP provided much cleaner extracts and improved analytical performance. Quantification of homocysteine, cysteine, and methionine was performed using their deuterated analogues, whereas other analytes were quantified by means of 4-chlorophenylalanine. Precise and reliable assay of all examined analytes was achieved, irrespective of the chloroformate reagent used. Average relative standard deviations at each analyte level were ≤6%, quantification limits were 0.1-0.2 µmol L(-1), recoveries were 94-121%, and linearity was over three orders of magnitude (r(2) equal to 0.997-0.998). Validation performed with the THP agent and propyl chloroformate derivatization demonstrated the robustness and reliability of this simple sample-preparation methodology.

Specific microRNAs and heart failure: time for the next step toward application?

A number of microRNAs are involved in the pathophysiological events associated with heart disease. In this review, we discuss miR-21, miR-1, miR-23a, miR-142-5p, miR-126, miR-29, miR-195, and miR-499 because they are most often mentioned as important specific indicators of myocardial hypertrophy and fibrosis leading to heart failure. The clinical use of microRNAs as biomarkers and for therapeutic interventions in cardiovascular diseases appears highly promising. However, there remain many unresolved details regarding their specific actions in distinct pathological phenomena. The introduction of microRNAs into routine practice, as part of the cardiovascular examination panel, will require additional clinically relevant and reliable data. Thus, there remains a need for additional research in this area, as well as the optimization and standardization of laboratory procedures which could significantly shorten the determination time, and make microRNA analysis simpler and more affordable. In this review, we aim to summarize the current knowledge about selected microRNAs related to heart failure, including their potential use in diagnosis, prognosis, and treatment, and options for their laboratory determination.

Breath Tests Used in the Context of Bariatric Surgery.

This review article focuses on the use of breath tests in the field of bariatrics and obesitology. The first part of the review is an introduction to breath test problematics with a focus on their use in bariatrics. The second part provides a brief history of breath testing. Part three describes how breath tests are used for monitoring certain processes in various organs and various substances in exhaled air and how the results are analyzed and evaluated. The last part covers studies that described the use of breath tests for monitoring patients that underwent bariatric treatments. Although the number of relevant studies is small, this review could promote the future use of breath testing in the context of bariatric treatments.

Methylene blue: a controversial diagnostic acid and medication?

A narrative review of the literature was conducted to determine if the administration of methylene blue (MB) in humans has potential risks. Studies were identified from MEDLINE, Web of Science, Scopus, and Cochrane. MB is a diagnostic substance used during some diagnostic procedures and also a part of the treatment of several diseases including methemoglobinemia, vasoplegic syndrome, fosfamide-induced encephalopathy, and cyanide intoxication, and the detection of leaks or position of parathyroid corpuscles during surgery. Although the use of MB is historically justified, and it ought to be safe, because it originated as a diagnostic material, the basic toxicological characteristics of this substance are unknown. Despite reports of severe adverse effects of MB, which could significantly exceed any possible benefits evaluated for the given indication. Therefore, the clinical use of MB currently represents a controversial problem given the heterogeneity of available data and the lack of preclinical data. This is in conflict with standards of safe use of such substances in human medicinal practice. The toxic effects of the application of MB are dose-dependent and include serious symptoms such as hemolysis, methemoglobinemia, nausea and vomitus, chest pain, dyspnoea, and hypertension. Some countries regard MB as harmful because of the resulting skin irritation and triggering of an adverse inflammatory response. MB induced serotoninergic toxicity clinically manifests as neuromuscular hyperactivity. This review aims to summarize the current understanding concerning the indications for MB administration and define the potential adverse effects of MB.

Secretoneurin as a Novel Biomarker of Cardiovascular Episodes: Are We There Yet? A Narrative Review.

Secretoneurin (SN) is a 33 amino-acid evolutionary conserved neuropeptide from the chromogranin peptide family. SN's main effects may be cardioprotective and are believed to be mediated through its inhibition of calmodulin-dependent kinase II (CaMKII), which influences intracellular calcium handling. SN inhibition of CaMKII suppresses calcium leakage from the sarcoplasmic reticulum through the ryanodine receptor. This action may reduce the risk of ventricular arrhythmias and calcium-dependent remodelling in heart failure. SN is also involved in reducing the intracellular reactive oxygen species concentration, modulating the immune response, and regulating the cell cycle, including apoptosis. SN can predict mortality in different disease states, beyond the classical risk factors and markers of myocardial injury. Plasma SN levels are elevated soon after an arrhythmogenic episode. In summary, SN is a novel biomarker with potential in cardiovascular medicine, and probably beyond.

Use of Magnetic Resonance Imaging to Quantify Fat and Steatosis in the Pancreas in Patients after Bariatric Surgery: a Retrospective Study.

INTRODUCTION: Pancreatic steatosis (PS) has both metabolic consequences and local effects on the pancreas itself. Magnetic resonance imaging (MRI) is the most reliable non-invasive method for diagnosing PS. We investigated the impact of metabolic syndrome (MS) on the presence of PS, differences in individuals with and without PS, and the metabolic effects of bariatric procedures. METHODS: Changes in anthropometric and basic biochemistry values and MS occurrence were evaluated in 34 patients with obesity who underwent a bariatric procedure. After the procedure, patients underwent MRI with manual 3D segmentation mask creation to determine the pancreatic fat content (PFC). We compared the differences in the PFC and the presence of PS in individuals with and without MS and compared patients with and without PS. RESULTS: We found no significant difference in the PFC between the groups with and without MS or in the occurrence of PS. There were significant differences in patients with and without PS, especially in body mass index (BMI), fat mass, visceral adipose tissue (VAT), select adipocytokines, and lipid spectrum with no difference in glycemia levels. Significant metabolic effects of bariatric procedures were observed. CONCLUSIONS: Bariatric procedures can be considered effective in the treatment of obesity, MS, and some of its components. Measuring PFC using MRI did not show any difference in relation to MS, but patients who lost weight to BMI < 30 did not suffer from PS and had lower overall fat mass and VAT. Glycemia levels did not have an impact on the presence of PS.

Five Antigen Tests for SARS-CoV-2: Virus Viability Matters.

Antigen testing for SARS-CoV-2 (AGT) is generally considered inferior to RT-PCR testing in terms of sensitivity. However, little is known about the infectiousness of RT-PCR positive patients who pass undetected by AGT. In a screening setting for mildly symptomatic or asymptomatic patients with high COVID-19 prevalence (30-40%), 1141 patients were tested using one of five AGTs and RT-PCR. Where the results differed, virus viability in the samples was tested on cell culture (CV-1 cells). The test battery included AGTs by JOYSBIO, Assure Tech, SD Biosensor, VivaChek Biotech and NDFOS. Sensitivities of the ATGs compared to RT-PCR ranged from 42% to 76%. The best test yielded a 76% sensitivity, 97% specificity, 92% positive, and 89% negative predictive values, respectively. However, in the best performing ATG tests, almost 90% of samples with "false negative" AGT results contained no viable virus. Corrected on the virus viability, sensitivities grew to 81-97% and, with one exception, the tests yielded high specificities >96%. Performance characteristics of the best test after adjustment were 96% sensitivity, 97% specificity, 92% positive, and 99% negative predictive values (high prevalence population). We, therefore, believe that virus viability should be considered when assessing the AGT performance. Also, our results indicate that a well-performing antigen test could in a high-prevalence setting serve as an excellent tool for identifying patients shedding viable virus. We also propose that the high proportion of RT-PCR-positive samples containing no viable virus in the group of "false negatives" of the antigen test should be further investigated with the aim of possibly preventing needless isolation of such patients.

Nutritional consequences of endoluminal gastroplasty in the treatment of morbidly obese patients.

BACKGROUND: The Endoscopic sleeve gastroplasty (ESG) is one of the new advanced restrictive techniques of bariatric endoscopy. The principle of this bariatric technique is the reduction of stomach volume through endoscopic plication. The long-term efficacy and metabolic effects of this procedure are the subject of this study. METHODS: 20 patients were enrolled; four men, sixteen women. Then they were followed for 24 months in terms of glycemic control, body composition, vitamin, and nutritional status. Observed parameters included glucose, triacylglycerols, high- and low-density lipoprotein cholesterol, micro and macro nutrients, fat mass, and lean body mass. RESULTS: We observed significant weight loss and a decrease body composition resulted from procedure vs. baseline, with levels of blood glucose also showing statistically significant reductions. The most notable decline in measured values was observed in period six months after the procedure. No significant changes were observed in measurement of micronutrients. CONCLUSIONS: Good restriction results were obtained following ESG, which might be mediated via altered glucose metabolism. The ESG method has shown a positive effect on fat and muscle mass. Unlike surgical methods, there were no deficits or deficiencies, especially in terms of essential vitamin levels.

Covid-19 antigen testing: better than we know? A test accuracy study.

BACKGROUND: Antigen testing for SARS-CoV-2 is considered to be less sensitive than the standard reference method - real-time PCR (RT-PCR). It has been suggested that many patients with positive RT-PCR 'missed' by antigen testing might be non-infectious. METHODS: In a real-world high-throughput setting for asymptomatic or mildly symptomatic patients, 494 patients were tested using RT-PCR as well as a single lateral flow antigen test (Ecotest, AssureTech, China). Where the results differed, virus viability was evaluated by cell culture. The test parameters were calculated with RT-PCR and RT-PCR adjusted on viability as reference standards. RESULTS: The overall sensitivity of the used antigen test related to the RT-PCR only was 76.2%, specificity was 97.3%. However, 36 out of 39 patients 'missed' by the antigen test contained no viable virus. After adjusting on that, the sensitivity grew to 97.7% and, more importantly for disease control purposes, the negative predictive value reached 99.2%. CONCLUSIONS: We propose that viability testing should be always performed when evaluating a new antigen test. A well-chosen and validated antigen test provides excellent results in identifying patients who are shedding viable virus (although some caveats still remain) in the real-world high-throughput setting of asymptomatic or mildly symptomatic individuals.

Measurement of novel adipokine visfatin in young patients with acute myocardial infarction. Clinical testing of a new ELISA.

OBJECTIVES: Adipose tissue produces a number of adipokines that have metabolic effect. Visfatin is a recently discovered adipokine whose concentration in plasma increases in obesity. It is also a proinflammatory mediator that promotes atherosclerosis and plays a role in plaque destabilization. The aim of this study was to evaluate an assay for the determination of visfatin in human plasma and to investigate its clinical relevance as a marker of acute coronary syndrome (ACS) in a young population (Men under 45 y, Women under 55 y). DESIGN AND METHODS: We clinically tested a sandwich ELISA assay in young individuals with acute myocardial infarction (n=36) vs. a control group (n=21). The control sample was a healthy proband without inflammation, hepatic or renal injury and under 55 years of age. RESULTS: Visfatin in plasma was able to differentiate the control group from young patients with acute myocardial infarction (5 vs. 27 ng/L). Visfatin in the plasma of acute myocardial infarction (AMI) probands, correlated in individuals with acute coronary syndrome was related to plasma glucose (r=0.47; P=0.01), type 2 diabetes mellitus (r=0.65; P=0.01), plasma creatinine concentration (r=0.3, P=0.02), hsCRP (r=0.29; P=0.03), BMI values (r=0.18; P=0.04), triglycerides (r=0.5; P=0.01) and NT-proBNP (r=0.21; P=0.04). In healthy subjects, these relations were not found. ROC analysis: visfatin cut-off concentration was 20 ng/L with a sensitivity of 84% and a specificity of 90%. The area under the curve (AUC) of cTNI was 0.96, the AUC of visfatin was 0.96. Thus, there was no difference. CONCLUSION: We conclude that visfatin in serum may be a new independent potential marker of AMI.

Changes in cholesterol metabolism during acute upper gastrointestinal bleeding: liver cirrhosis and non cirrhosis compared.

BACKGROUND: Cholesterol is derived via de novo synthesis and dietary absorption. Both processes can be monitored by determination of non-cholesterol sterol concentrations (lathosterol for synthesis; sitosterol and campesterol for absorption). The hypocholesterolemia that occurs during acute illness is a result of a multifactorial inability to compensate for the increased needs for this metabolite. The aim of this study was to examine the plasma cholesterol profile and both processes of cholesterol acquisition during acute upper gastrointestinal haemorrhage with emphasis on liver cirrhosis. MATERIAL AND METHODS: Thirty five patients with acute upper gastrointestinal bleeding (cirrhosis n=14, non-cirrhosis n=21) were evaluated over a 6 day period. The control cohort consisted of 100 blood donors. Serum concentrations of total, LDL (low-density lipoprotein) and HDL (high-density lipoprotein) cholesterol were measured enzymatically. Sterol concentrations were analysed using gas chromatography, data were statistically analysed. RESULTS: In all patients, we found lower plasma levels of total cholesterol (P Conclusion: Our results showed substantial abnormalities in the cholesterol plasma profile including both the processes of cholesterol acquisition in patients with upper acute gastrointestinal bleeding. The patients with or without liver cirrhosis had similar trends in cholesterol plasma levels. Depression of cholesterol synthesis was, however, prolonged in the cirrhotic group and the data also suggest a different phytosterol metabolism.

Comparison of a new immunoassay and PCR-based method for quantification of microRNAs in whole blood. A pilot methodical study.

BACKGROUND: MicroRNAs (miRNAs) are new generation biomarkers used in oncology, cardiology, metabolic syndrome, obesity or in neurology. miRNAs are short non-coding RNA molecules that regulate gene expression in eukaryotes. AIM: To compare a new commercial method for establishing miRNA (imunoassay) with a commercial kit RT qPCR. METHODS: RNA was isolated from whole blood samples obtained from four healthy volunteers. The isolates were liquated and miRNA-93-5p and miRNA-23a-3p were measured independently with commercial hsa-miR-93-5p miREIA and hsa-miR-23a-3p miREIA, and commercial RT-qPCR kits. RESULTS: Both miRNAs had good analytical characteristics, very good correlation with RT qPCR. The results between immunoassay and RT qPCR did not statistically differ. A method based on ELISA was faster (2 h with ELISA vs. 3 h with qPCR) and had lower CV then a method based on RT qPCR (see more text). CONCLUSION: MicroRNAs from blood or derived fractions are particularly interesting candidates for routine laboratory applications. The immunoassay can be performed on any device that processes the ELISA plates and is therefore available in almost every laboratory.

GC-MS Metabolomic Profiling of Protic Metabolites Following Heptafluorobutyl Chloroformate Mediated Dispersive Liquid Microextraction Sample Preparation Protocol.

A simple analytical workflow is described for gas chromatographic-mass spectrometry (GC-MS)-based metabolomic profiling of protic metabolites, particularly amino-carboxylic species in biological matrices. The sample preparation is carried out directly in aqueous samples and uses simultaneous in situ heptafluorobutyl chloroformate (HFBCF) derivatization and dispersive liquid-liquid microextraction (DLLME), followed by GC-MS analysis in single-ion monitoring (SIM) mode. The protocol involves ten simple pipetting steps and provides quantitative analysis of 132 metabolites by using two internal standards. A comment on each analytical step and explaining notes are provided with particular attention to the GC-MS analysis of 112 physiological metabolites in human urine.