Metabolic Evaluation of Urine from Patients Diagnosed with High Grade (HG) Bladder Cancer by SPME-LC-MS Method.

Bladder cancer (BC) is a common malignancy of the urinary system and a leading cause of death worldwide. In this work, untargeted metabolomic profiling of biological fluids is presented as a non-invasive tool for bladder cancer biomarker discovery as a first step towards developing superior methods for detection, treatment, and prevention well as to further our current understanding of this disease. In this study, urine samples from 24 healthy volunteers and 24 BC patients were subjected to metabolomic profiling using high throughput solid-phase microextraction (SPME) in thin-film format and reversed-phase high-performance liquid chromatography coupled with a Q Exactive Focus Orbitrap mass spectrometer. The chemometric analysis enabled the selection of metabolites contributing to the observed separation of BC patients from the control group. Relevant differences were demonstrated for phenylalanine metabolism compounds, i.e., benzoic acid, hippuric acid, and 4-hydroxycinnamic acid. Furthermore, compounds involved in the metabolism of histidine, beta-alanine, and glycerophospholipids were also identified. Thin-film SPME can be efficiently used as an alternative approach to other traditional urine sample preparation methods, demonstrating the SPME technique as a simple and efficient tool for urinary metabolomics research. Moreover, this study's results may support a better understanding of bladder cancer development and progression mechanisms.

Elimination Characteristics of the Alcohol Biomarker Phosphatidylethanol (PEth) in Blood during Alcohol Detoxification.

AIMS: The study documented elimination characteristics of three phosphatidylethanol (PEth) homologs in serially collected blood samples from 47 heavy drinkers during ~2 weeks of alcohol detoxification at hospital. METHODS: Venous whole blood and urine samples were collected every 1-2 days during treatment. Concentrations of PEth, and of urinary ethyl glucuronide (EtG) and ethyl sulfate (EtS) to detect relapse drinking, were measured using liquid chromatography-tandem mass spectrometry. RESULTS: When included in the study, negative or decreasing breath ethanol concentrations demonstrated that the patients were in the elimination phase. The EtG and EtS measurements further confirmed alcohol abstinence during the study, with three exceptions. On admission, all patients tested positive for PEth, the total concentration ranging 0.82-11.7 (mean 6.35, median 5.88) µmol/l. PEth 16:0/18:1, 16:0/18:2 and 16:0/20:4 accounted for on average ~42%, ~26% and ~9%, respectively, of total PEth in these samples. There were good correlations between total PEth and individual homologs (P < 0.0001). There was no significant difference in PEth values between male and female subjects. During abstinence, the elimination half-life values ranged 3.5-9.8 days for total PEth, 3.7-10.4 days for PEth 16:0/18:1, 2.7-8.5 days for PEth 16:0/18:2 and 2.3-8.4 days for PEth 16:0/20:4. CONCLUSIONS: The results demonstrated a very high sensitivity (100%) of PEth as alcohol biomarker for recent heavy drinking, but considerable differences in the elimination rates between individuals and between different PEth forms. This indicates that it is possible to make only approximate estimates of the quantity and recency of alcohol intake based on a single PEth value.

Improved detection of alcohol consumption using the novel marker phosphatidylethanol in the transplant setting: results of a prospective study.

Phosphatidylethanol (PEth) is a new, highly specific alcohol marker. The aim of this study was to assess its diagnostic value in the liver transplant setting. In 51 pre- and 61 post-transplant patients with underlying alcoholic liver disease PEth, ethanol, methanol, carbohydrate-deficient transferrin (CDT), and ethyl glucuronide in urine (uEtG) and hair (hEtG) were tested and compared with patients' questionnaire reports. Twenty-eight (25%) patients tested positive for at least one alcohol marker. PEth alone revealed alcohol consumption in 18% of patients. With respect to detection of alcohol intake in the preceding week, PEth showed a 100% sensitivity. PEth testing was more sensitive than the determination of ethanol, methanol, CDT or uEtG alone [sensitivity 25% (confidence interval (CI) 95%, 7-52%), 25% (7-52%), 21% (6-45%) and 71% (41-91%), respectively], or ethanol, methanol and uEtG taken in combination with 73% (45-92%). Specificity of all markers was 92% or higher. Additional testing of hEtG revealed alcohol consumption in seven patients, not being positive for any other marker. Phosphatidylethanol was a highly specific and sensitive marker for detection of recent alcohol consumption in pre- and post-transplant patients. The additional determination of hEtG was useful in disclosing alcohol consumption 3-6 months retrospectively.

Phosphatidylethanol (PEth) detected in blood for 3 to 12 days after single consumption of alcohol-a drinking study with 16 volunteers.

In most studies, the alcohol marker phosphatidylethanol (PEth) was used to differentiate social drinking from alcohol abuse. This study investigates PEth's potential in abstinence monitoring by performing a drinking study to assess the detection window of PEth after ingesting a defined amount of alcohol. After 2 weeks of abstinence, 16 volunteers ingested a single dose of alcohol, leading to an estimated blood alcohol concentration (BAC) of 1 g/kg. In the week after drinking, blood and urine samples were taken daily; in the second week, samples were taken every other day. PEth 16:0/18:1 and 16:0/18:2 were analyzed in blood by online-SPE-LC-MS/MS. Ethyl glucuronide and ethyl sulfate were determined in urine for abstinence monitoring. Prior to start of drinking, PEth 16:0/18:1 exceeded 30 ng/mL in blood samples of five volunteers despite the requested abstinence period. Positive PEth values resulted from drinking events prior to this abstinence period. After the start of drinking, maximum BACs were reached after 2 h with a mean of 0.80 ± 0.13 g/kg (range: 0.61-1.11 g/kg). PEth 16:0/18:1 increased within 8 h to maximum concentrations (mean: 88.8 ± 47.0 ng/mL, range: 37.2-208 ng/mL). After this event, PEth was detectable for 3 to 12 days with a mean half-life time of approximately 3 days. PEth has a potential in abstinence monitoring, since PEth could be detected for up to 12 days after a single drinking event. Further investigations are necessary, to establish cut-off levels for PEth as diagnostic marker for the determination of drinking habits like abstinence, social drinking, or risky alcohol consumption.

[Tests for alcohol consumption during pregnancy: what biomarkers are suitable?]. / Testen op alcoholgebruik in de zwangerschap.

Alcohol consumption during pregnancy may lead to severe foetal damage, such as foetal alcohol spectrum disorders. It is known that pregnant women under-report to questionnaires about alcohol consumption. It is therefore necessary to determine alcohol consumption during pregnancy objectively. We present 2 pregnant women with negative urine tests for ethyl glucuronide (EtG) and alcohol. However, analysis of two other biomarkers, phosphatidylethanol (PEth) in blood and fatty acid ethyl esters (FAEE) in meconium, revealed alcohol consumption during pregnancy by both women. Analysis of PEth can yield additional information alongside EtG testing. This is due to the much longer half-life of PEth. Meconium testing for FAEE provides relevant information about alcohol consumption during the second and third trimesters. Both PEth and meconium analysis can help identify women who have consumed alcohol during pregnancy. Appropriate counselling and follow-up can be given to these mothers and their children.

Determination of direct alcohol markers: a review.

Alcohol is the most popular legal drug used in our society today, and its consumption by pregnant women remains an important public health problem. Gestational alcohol consumption can result in a continuum of adverse fetal outcomes known as fetal alcohol spectrum disorder (FASD). Effective strategies are needed to prevent the increasing adoption of risky drinking behaviors. Because ethanol itself is only measurable for a few hours after ethanol intake in conventional matrices including blood, urine, and sweat, these matrices are only useful to detect recent ethanol exposure. Since approximately early 2000, the non-oxidative ethanol metabolites have received increasing attention because of their specificity and, in some cases, wide time window of detection in non-conventional matrices including hair and meconium. In the attempt to update analytical methods for the determination of non-oxidative markers of alcohol, the objective of this study is to review published studies that measure fatty-acid ethyl esters (FAEE), ethyl glucuronide (EtG), and phosphatidylethanol (PEth) in alternative biological matrices, focusing on the extraction and detection methods and full analytical conditions used.

Phosphatidylethanol: the potential role in further evaluating low positive urinary ethyl glucuronide and ethyl sulfate results.

BACKGROUND: Whereas urinary ethyl glucuronide (EtG) levels above 1,000 ng/ml reflect with a high probability ethanol (EtOH) consumption, levels below this cutoff are difficult to interpret as both extraneous (nonbeverage) EtOH exposure, recent drinking, and more distant high EtOH intake (several days ago) might yield similar results. This might be of particular relevance in medico-legal cases. To overcome this dilemma, phosphatidylethanol (PEth) might be a promising marker, because blood PEth is only positive following significant alcohol use. The aim of our study was therefore to employ PEth as a marker to differentiate between the different conditions. METHODS: Subjects included were 252 participants in monitoring with the Alabama Physician Health Program. All subjects testing positive for EtG and/or ethyl sulfate (EtS) who denied drinking after routine supportive confrontation were subject to information about PEth testing. If they still denied drinking, PEth testing was performed and the result communicated. EtG, EtS, and PEth testing was performed in a commercial laboratory using liquid chromatography tandem mass spectrometry methods. RESULTS: Of a total of 18 subjects who tested positive for EtG and/or EtS, 10 denied drinking. Of the 7 who denied drinking after PEth explanation, in 5 cases, their claim was supported by a negative PEth result. In 2 cases, a positive PEth result was in contrast to their claim. CONCLUSIONS: PEth results in combination with previous low positive EtG/EtS results allow differentiating between innocent/extraneous exposure and drinking. Negative PEth testing following low positive EtG/EtS results helps to further elucidate the findings and support the claim of the patient of recent alcohol abstinence. Positive PEth testing following positive EtG/EtS results confirms recent drinking.

Metabolomic study of biochemical changes in the plasma and urine of primary dysmenorrhea patients using UPLC-MS coupled with a pattern recognition approach.

Primary dysmenorrhea (PD) is characterized by painful menstrual cramps without any organic pathology and has a prevalence of up to 90% in adolescents. Recent advances in its etiology and pathogenesis are providing more speculative hypotheses focused on integral systems. Using a targeted tandem mass spectrometry (MS/MS)-based metabolomic platform, we explored the changes of metabolic profiling in plasma/urine simultaneously between PD patients and healthy controls before and after a 3-month herbal medicine (namely Shaofu Zhuyu formula concentrated-granule, SFZYFG) therapy. To detect and identify potential biomarkers associated with PD and SFZYFG treatment, we also performed a combined UPLC-QTOF-MS/MS-based metabolomic profiling of the plasma/urine samples, indicating a further deviation of the patients' global metabolic profile from that of controls. The total thirty-five metabolites (nineteen in plasma and sixteen in urine), up-regulated or down-regulated (p < 0.05 or 0.01), were identified and contributed to PD progress. These promising identified biomarkers underpinning the metabolic pathway including sphingolipid metabolism, steroid hormone biosynthesis, and glycerophospholipid metabolism are disturbed in PD patients, which were identified by using pathway analysis with MetPA. Twenty-four altered metabolites and fourteen biochemical indicators were restored back to the control-like level after the treatment of SFZYFG and could be potential biomarkers for monitoring therapeutic efficacy. These findings may be promising to yield a valuable insight into the pathophysiology of PD and to advance the approaches of treatment, diagnosis, and prevention of PD and related syndromes.

Comparison of direct and indirect alcohol markers with PEth in blood and urine in alcohol dependent inpatients during detoxication.

The importance of direct and indirect alcohol markers to evaluate alcohol consumption in clinical and forensic settings is increasingly recognized. While some markers are used to prove abstinence from ethanol, other markers are suitable for detection of alcohol misuse. Phosphatidyl ethanol (PEth) is ranked among the latter. There is only little information about the correlation between PEth and other currently used markers (ethyl glucuronide, ethyl sulfate, carbohydrate deficient transferrin, gamma-glutamyl transpeptidase, and methanol) and about their decline during detoxification. To get more information, 18 alcohol-dependent patients in withdrawal therapy were monitored for these parameters in blood and urine for up to 19 days. There was no correlation between the different markers. PEth showed a rapid decrease at the beginning of the intervention, a slow decline after the first few days, and could still be detected after 19 days of abstinence from ethanol.

A hyphenated microLC-Q-TOF-MS platform for exosomal lipidomics investigations: application to RCC urinary exosomes.

Urinary exosomes are released from every renal epithelial cell type facing the urinary space and therefore, they may carry molecular markers of renal dysfunction and structural injury. Here, we present a hyphenated microLC-Q-TOF-MS platform for lipidomics studies applied to investigate the urinary exosome lipid repertoire. Lipids were separated by reversed-phase chromatography using a linear gradient of formic acid 0.2% and tetrahydrofuran, in 40 min of analysis. Features (m/z with associated own retention time) were extracted by MarkerLynx(TM) (Waters) and processed, demonstrating good analytical performance in terms of repeatability and mass accuracy of the microLC Q-TOF MS platform. In particular, a stable retention time (RSD less than 4%) and relative intensity (RSD from 2.9% to 11%) were observed. Moreover, the method takes advantages by the use of a lock spray interface (Waters) that allows readjusting the m/z data after acquisition, obtaining inaccuracy below 6 ppm in measuring the m/z value of the reference compound during chromatographic run. The method was employed in a preliminary application to perform comparative analysis from healthy control subjects and renal cell carcinoma (RCC) patients, in order to possibly highlight differences in lipid composition to be exploited as potential tumor biomarker. Differential lipid composition in RCC urinary exosomes was achieved and tentatively identified by accurate mass, providing a preliminary indication of a relationship between lipid composition of urinary exosomes and RCC disease. Among the total features significantly different in RCC exosomes, the ion at m/z 502.3 was taken as an example for molecular confirmation by MS/MS fragmentation analysis.

Ethyl glucuronide discloses recent covert alcohol use not detected by standard testing in forensic psychiatric inpatients.

BACKGROUND: Considerable lives and money could be saved if one could detect early stages of lapsing/relapsing behavior in addicted persons (e.g., in safety-sensitive workplaces) and could disclose harmful drinking in social drinkers. Due to the serious public health problem of alcohol use and abuse worldwide, markers of alcohol use have been sought. Both ethyl glucuronide (EtG) and phosphatidyl ethanol (PEth) appear to have high sensitivity and specificity and a time frame of detection that may elucidate alcohol use not detected by standard testing. Our aim was to assess their potential for detecting recent covert alcohol use under controlled conditions. METHODS: Thirty-five forensic psychiatric inpatients in a closed ward who had committed a substance-related offense ( section sign 64 StGB), were followed for 12 months. The complete time spectrum of possible alcohol consumption was covered by the complementary use of breath and urinary ethanol (hours), urinary EtG (days), %carbohydrate-deficient transferrin (CDT)/PEth (weeks), and gamma-glutamyltranspeptidase (GGT)/mean corpuscular volume (MCV) (weeks-months). RESULTS: Fourteen of the 146 urine samples examined were positive for EtG. In all EtG-positive cases, patients reported alcohol consumption of between 40 and 200 g of ethanol 12-60 hr prior to testing. Urinary and breath ethanol were positive in only one case. In the blood samples, PEth was not positive in any case and %CDT did not exceed the reference value. Isoelectric focusing showed no abnormal Tf subtypes. CONCLUSIONS: The findings emphasize the diagnostic and therapeutic usefulness, specificity, and sensitivity of EtG as a marker of recent alcohol use. Such a test is needed in numerous settings, including alcohol and drug treatment (to detect lapse/relapse), in safety-sensitive work settings where use is dangerous or in other settings where use may be inappropriate (e.g., such as driving, workplace, pregnancy, or monitoring physicians or other professionals who are in recovery and working), or for testing other groups (such as children or those with medical problems) where alcohol use would be unhealthy or unsafe. The health, social and socioeconomic benefits arising from the future use of these markers is hard to overestimate.