Metabolomics analysis of urine from patients with alcohol-associated liver disease reveals dysregulated caffeine metabolism.

Excess alcohol intake causes millions of deaths annually worldwide. Asymptomatic early-stage, alcohol-associated liver disease (ALD) is easily overlooked, and ALD is usually only diagnosed in more advanced stages. We explored the possibility of using polar urine metabolites as biomarkers of ALD for early-stage diagnosis and functional assessment of disease severity by quantifying the abundance of polar metabolites in the urine samples of healthy controls (n = 18), patients with mild or moderate liver injury (n = 21), and patients with severe alcohol-associated hepatitis (n = 25). The polar metabolites in human urine were first analyzed by untargeted metabolomics, showing that 209 urine metabolites are significantly changed in patients, and 17 of these are highly correlated with patients' model for end-stage liver disease (MELD) score. Pathway enrichment analysis reveals that the caffeine metabolic pathway is the most affected in ALD. We then developed a targeted metabolomics method and measured the concentration of caffeine and its metabolites in urine using internal and external standard calibration, respectively. The described method can quantify caffeine and its 14 metabolites in 35 min. The results of targeted metabolomics analysis agree with the results of untargeted metabolomics, showing that 13 caffeine metabolites are significantly decreased in patients. In particular, the concentrations of 1-methylxanthine, paraxanthine, and 5-acetylamino-6-amino-3-methyluracil are markedly decreased with increased disease severity. We suggest that these three metabolites could serve as functional biomarkers for differentiating early-stage ALD from more advanced liver injury.NEW & NOTEWORTHY Our study using both untargeted and targeted metabolomics reveals the caffeine metabolic pathway is dysregulated in ALD. Three caffeine metabolites, 1-methylxanthine, paraxanthine, and 5-acetylamino-6-amino-3-methyluracil, can differentiate the severity of early-stage ALD.

Could protein content of Urinary Extracellular Vesicles be useful to detect Cirrhosis in Alcoholic Liver Disease?

Alcohol abuse has a high impact on the mortality and morbidity related to a great number of diseases and is responsible for the development of alcoholic liver disease (ALD). It remains challenging to detect and evaluate its severity, which is crucial for prognosis. In this work, we studied if urinary EVs (uEVs) could serve in diagnose and evaluate cirrhosis in ALD. To this purpose, uEVs characterization by cryo-electron microscopy (Cryo-EM), Nanoparticle Tracking Analysis (NTA) and Western blotting (WB) was performed in a cohort of 21 controls and 21 cirrhotic patients. Then, proteomics of uEVs was carried out in a second cohort of 6 controls and 8 patients in order to identify new putative biomarkers for cirrhosis in ALD. Interestingly, uEVs concentration, size and protein composition were altered in cirrhotic patients. From a total of 1304 proteins identified in uEVs, 90 of them were found to be altered in cirrhotic patients. The results suggest that uEVs could be considered as a tool and a supplier of new biomarkers for cirrhosis in ALD, whose application would be especially relevant in chronic patients. Yet, further research is necessary to obtain more relevant result in clinical terms.

Role of Alcohol and Drug Detection by Regular Urine Sample Testing in pre-transplant evaluation for Alcohol Liver Disease. / Papel de la detección regular de sustancias en orina en pacientes en valoración pre-transplante hepático por hepatopatía alcohólica.

Alcohol Liver Disease (ALD) is one of the most prevalent conditions leading to liver transplantation for end-stage liver disease. There is lacking evidence of regular urine screening testing (RUST) impact on survival or liver transplantation of ALD patients. The aims of this study were to compare the sensitivity of RUST, to assess its impact on survival and liver transplantation, and to evaluate factors associated with adherence to RUST. We performed a single-centered retrospective study (N = 84) with ALD candidates for liver transplantation. Demographic, biochemical and clinical variables were recorded at baseline. Adherence to RUST was evaluated during follow-up. The sensitivity of both RUST and self-reports were calculated for all drugs. Multivariable logistic and survival regression analyses were performed to explore associated factors and the impact of adherence to RUST, and positive results on survival. RUST had high sensitivity for identifying active drinkers (76.9%), smokers (78.9%) and cannabis users (83.3%). High adherence to RUST was inversely associated with mortality during follow-up. Presence of personality disorders negatively impacted (OR 0.29, CI 95% 0.08-0.97) adherence to RUST. Both RUST and self-reports should be carried out in this setting. Professionals involved in liver transplantation programs must promote adherence to RUST, primarily in patients with personality disorders.

La enfermedad hepática alcohólica (EHA) es una de las causas más frecuentes de trasplante hepático en enfermedad hepática terminal. No hay evidencia de impacto de la detección regular de sustancias en orina (DRSO) sobre la supervivencia de los pacientes con EHA. Los objetivos de este estudio fueron comparar la sensibilidad de la DRSO, evaluar su impacto en la supervivencia y en el trasplante de hígado, y evaluar el impacto de la adherencia a la DRSO. Realizamos un estudio retrospectivo (N = 84) con candidatos para trasplante de hígado por EHA. Registramos las variables demográficas, bioquímicas y clínicas al inicio del estudio. Evaluamos la adherencia a la DRSO durante el seguimiento. Calculamos la sensibilidad tanto de la DRSO como de las declaraciones de los pacientes para todas las sustancias. Realizamos análisis multivariables (regresión logística) y de supervivencia para explorar los factores asociados y el impacto de la adherencia a la DRSO, y de los resultados positivos en la DRSO sobre la supervivencia. La DRSO tuvo una alta sensibilidad para identificar bebedores activos (76,9%), fumadores (78,9%) y consumidores de cannabis (83,3%). Alta adherencia a la DRSO tuvo una asociación inversa con mortalidad durante el seguimiento. La presencia de trastornos de la personalidad tuvo un impacto negativo (RM ,29, IC 95% ,08-,97) sobre la adherencia a la DRSO. Tanto la DRSO como las declaraciones deben llevarse a cabo en este perfil de pacientes. Los profesionales que participan en programas de trasplante hepático deben promover el cumplimiento de la DRSO, principalmente en pacientes con trastornos de la personalidad.

Papel de la detección regular de sustancias en orina en pacientes en valoración pre-transplante hepático por hepatopatía alcohólica / Role of Alcohol and Drug Detection by Regular Urine Sample Testing in pre-transplant evaluation for Alcohol Liver Disease

La enfermedad hepática alcohólica (EHA) es una de las causas más frecuentes de trasplante hepático en enfermedad hepática terminal. No hay evidencia del impacto de la detección regular de sustancias en orina (DRSO) sobre la supervivencia de los pacientes con EHA. Los objetivos de este estudio fueron comparar la sensibilidad de la DRSO, evaluar su impacto en la supervivencia y en el trasplante hepático, y evaluar el impacto de la adherencia a la DRSO. Realizamos un estudio retrospectivo (N = 84) con candidatos para trasplante hepático por EHA. Registramos las variables demográficas, bioquímicas y clínicas al inicio del estudio. Evaluamos la adherencia a la DRSO durante el seguimiento. Calculamos la sensibilidad tanto de la DRSO como de las declaraciones de los pacientes para todas las sustancias. Realizamos análisis multivariables (regresión logística) y de supervivencia para explorar los factores asociados y el impacto de la adherencia a la DRSO, y de los resultados positivos en la DRSO sobre la supervivencia. La DRSO tuvo una alta sensibilidad para identificar bebedores activos (76,9%), fumadores (78,9%) y consumidores de cannabis (83,3%). La alta adherencia a la DRSO tuvo una asociación inversa con la mortalidad durante el seguimiento. La presencia de trastornos de la personalidad tuvo un impacto negativo (RM ,29, IC 95% ,08-,97) sobre la adherencia a la DRSO. Tanto la DRSO como las declaraciones deben llevarse a cabo en este perfil de pacientes. Los profesionales que participan en programas de trasplante hepático deben promover el cumplimiento de la DRSO, principalmente en pacientes con trastornos de la personalidad

Alcohol Liver Disease (ALD) is one of the most prevalent conditions leading to liver transplantation for end-stage liver disease. There is lacking evidence of regular urine screening testing (RUST) impact on survival or liver transplantation of ALD patients. The aims of this study were to compare the sensitivity of RUST, to assess its impact on survival and liver transplantation, and to evaluate factors associated with adherence to RUST. We performed a single-centered retrospective study (N = 84) with ALD candidates for liver transplantation. Demographic, biochemical and clinical variables were recorded at baseline. Adherence to RUST was evaluated during follow-up. The sensitivity of both RUST and self-reports were calculated for all drugs. Multivariable logistic and survival regression analyses were performed to explore associated factors and the impact of adherence to RUST, and positive results on survival. RUST had high sensitivity for identifying active drinkers (76.9%), smokers (78.9%) and cannabis users (83.3%). High adherence to RUST was inversely associated with mortality during follow-up. Presence of personality disorders negatively impacted (OR 0.29, CI 95% 0.08-0.97) adherence to RUST. Both RUST and self-reports should be carried out in this setting. Professionals involved in liver transplantation programs must promote adherence to RUST, primarily in patients with personality disorders

Urinary acrolein metabolite levels in severe acute alcoholic hepatitis patients.

Alcohol-associated liver disease (ALD) remains a major health concern worldwide. Alcohol consumption gives rise to reactive/toxic acrolein, a pathogenic mediator of liver injury in experimental ALD. Elevated acrolein adducts and metabolites are detectable in blood and urine. This study evaluates the major urinary acrolein metabolite, 3-hydroxypropylmercapturic acid (HPMA), in patients with acute alcoholic hepatitis (AAH) and examines its association with disease severity and markers of hepatic inflammation and injury. Urine HPMA was significantly higher in patients with severe [model for end-stage liver disease (MELD) ≥ 20] AAH compared with nonsevere AAH (MELD ≤ 19) or non-alcohol-consuming controls, suggesting that urine HPMA is a novel noninvasive biomarker in severe AAH. The association between HPMA and MELD in patients with AAH was nonlinear. In patients with nonsevere AAH, there was a positive trend, although not significant, whereas in severe AAH the association was negative, indicative of extensive injury and glutathione depletion. Consistent with the multifactorial etiology of ALD, our data identified strong combined effects of HPMA and proinflammatory cytokines on hepatocyte cell death, thereby supporting the pathogenic role of acrolein in liver injury. HPMA, together with IL-1ß, showed robust associations with cytokeratin 18 caspase-cleaved fragment (CK18-M30; adjusted R2 = 0.812, P = 0.016) and cytokeratin 18 full-length protein (CK18-M65; adjusted R2 = 0.670, P = 0.048); similarly, HPMA, with IL-8, correlated with CK18-M30 (adjusted R2 = 0.875, P = 0.007) and CK18-M65 (adjusted R2 = 0.831, P = 0.013). The apoptosis index (CK18-M30:CK18-M65 ratio) strongly correlated with HPMA, together with IL-1ß (adjusted R2 = 0.777, P = 0.022) or tumor necrosis factor-α (TNFα; adjusted R2 = 0.677, P = 0.046). In patients with severe AAH, IL-1ß, IL-8, and TNFα are the predominant proinflammatory cytokines that interact with HPMA and play important mediating roles in influencing the extent/pattern of liver cell death. NEW & NOTEWORTHY This is the first study to examine the urinary acrolein metabolite 3-hydroxypropylmercapturic acid (HPMA) in alcoholic liver disease. HPMA was higher in patients with severe acute alcoholic hepatitis (AAH) compared with controls or nonsevere AAH and may be a novel selective, noninvasive biomarker for severe AAH. Consistent with the multifactorial etiology of alcohol-associated liver disease, we identified strong combined effects of HPMA and proinflammatory cytokines (IL-1ß, IL-8, and TNFα) on the extent/pattern of liver cell death, thereby supporting the pathogenic role of acrolein.

What's in Metabolomics for Alcoholic Liver Disease?

BACKGROUND AND AIMS: Current management of alcoholic liver disease (ALD), especially for alcoholic hepatitis (AH) is still driven by liver biopsy. Therefore, the identification of novel and accurate noninvasive biomarkers for the diagnosis and assessment of severity is important. Metabolomics, because it unravels changes closest to the phenotype, may represent the key for novel biomarkers. The aim of this study was to identify and characterize potential metabolomic biomarkers for diagnosis, staging and severity assessment of ALD. METHODS: 30 consecutive ALD patients and 10 healthy controls were included in this proof-of-concept cross-sectional study. Baseline assessment consisted in evaluation of Maddrey's Discriminant Function, Model for End-Stage Liver Disease (MELD) and ABIC scores as well as ASH-Test (Fibromax) as a surrogate for the confirmatory diagnosis of AH in suggestive clinical and biologic settings. Additionally, SOP metabolomics and lipidomics were performed from serum samples by liquid chromatography mass-spectrometry analysis. RESULTS: From the 127 and 135 serum/urine candidate metabolites initially identified, only 11/5 metabolites were characteristic for ALD patients. None of them correlated with alcohol intake, and only 5/1 metabolites could differentiate cirrhotic from non-cirrhotic patients. Of those, N-Lauroglycine (NLG) was the best for identifying cirrhosis (100% sensitivity and 90% negative predictive value, NPV) and decatrienoic acid (DTEA) was the best for assessing disease severity (evaluated by ABIC score) with 100% sensitivity and 100% NPV. CONCLUSION: Due to their high NPV, NLG and DTEA could be used in conjunction in ALD patients to exclude cirrhosis or a severe disease. If further validated, they could become biomarkers for better management and risk assessment in ALD.

Text Messaging to Reduce Alcohol Relapse in Prelisting Liver Transplant Candidates: A Pilot Feasibility Study.

BACKGROUND: Many liver transplantation programs require documented alcohol sobriety prior to United Network for Organ Sharing (UNOS) listing. This pilot study examined the feasibility of the first mobile, alcohol relapse prevention intervention for liver transplant patients with alcoholic liver disease (ALD). METHODS: This was a randomized 8-week pilot feasibility trial of a text message-based alcohol intervention. In-treatment assessment was conducted at 4 weeks (4W), and immediate posttreatment assessment was conducted at 8W. Participants were liver transplant candidates (N = 15) diagnosed with ALD who reported at least 1 drinking episode in the past year. Primary feasibility outcomes were percent of messages responded to and posttreatment intervention satisfaction ratings. Preliminary clinical efficacy outcomes were any biologically confirmed alcohol consumption, stress, abstinence self-efficacy, and alcohol craving. RESULTS: On feasibility outcomes, participants responded to 81% of messages received and reported high rates of intervention satisfaction, looked forward to receiving the messages, and found it easy to complete the intervention. On preliminary efficacy outcomes, zero participants in the text message (TM) had positive urine alcohol tests at 8W. Two of the 6 participants in standard care (SC) tested positive at 8W. No effects were seen on craving. For stress, a condition × time interaction emerged. TM participants had less stress at 4W and 8W compared with SC at baseline. They maintained their stress level during the intervention. For self-efficacy, a trend for condition effect emerged. TM participants had higher self-efficacy than SC participants. CONCLUSIONS: Participants reported high satisfaction with the intervention, looked forward to the messages, and found it easy to complete. Participants who received the intervention had better treatment outcomes than those who received standard care. They maintained higher levels of self-efficacy and lower stress. Mobile alcohol interventions may hold significant promise to help ALD liver transplant patients maintain sobriety.

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.

Dynamic metabolic profiling of urine biomarkers in rats with alcohol­induced liver damage following treatment with Zhi­Zi­Da­Huang decoction.

Alcoholic liver disease (ALD) is a leading cause of liver­associated morbidity and mortality. Zhi­Zi­Da­Huang decoction (ZZDHD), a traditional Chinese medicine formula, has been frequently used to treat or alleviate the symptoms of the various stages of ALD. To identify metabolic changes and the ZZDHD mechanism of action on ALD, potential urine biomarkers involved in the effects of ZZDHD were identified. Additionally, dynamic metabolomic profiles were systematically analyzed using nuclear magnetic resonance (NMR) spectroscopy in conjunction with statistical analysis. Alcohol administration to experimental rats disrupted multiple metabolic pathways, including methionine, gut bacterial, energy and amino acid metabolism. However, ZZDHD relieved certain effects of alcohol on the metabolism and regulated changes in potential characteristic biomarkers, including dimethylglycine, hippurate, lactate and creatine. The present study investigated time­dependent metabolomic changes in the development of alcohol­induced liver injury, including the effect of ZZDHD intervention. These findings elucidated important information regarding the metabolic responses to the protective effects of ZZDHD. 1H NMR­based metabolomics method a reliable and useful tool for determining the metabolic progression of alcohol­induced liver injury and elucidating the underlying mechanisms of the effect of traditional Chinese medicine formulas. This study also demonstrated that NMR­based metabolomics approach is a powerful tool for understanding the molecular basis of pathogenesis and drug intervention processes.

High resolution metabolomics technology reveals widespread pathway changes of alcoholic liver disease.

Alcoholic liver disease (ALD) is a significant cause of death and morbidity. However little is known regarding the widespread pathway changes of ALD disorder. This study utilized metabolomic profiling to examine the pathogenic mechanisms of ALD based on a rat model. A total of 21 metabolites with significant changes were identified, involving several key metabolic pathways such as pentose and glucuronate interconversions, starch and sucrose metabolism, cysteine and methionine metabolism. Furthermore, the differential proteins corresponding to alterations in metabolism across the metabolic network were identified using iTRAQ-based quantitative proteomics analysis. The proteins appear to be involved in protein binding, metabolism, immune response, and signal conduction. Interestingly, integrated omics profiling firstly reveals that p53 and Fc epsilon RI signaling pathways were closely related to ALD. Our study indicates that most of these proteins were found to play a pivotal role in the regulation of multiple metabolism pathways. Collectively, the current study provides insights into the molecular mechanisms of ALD from widespread pathway changes.

Urinary metabolic profiling of rat models revealed protective function of scoparone against alcohol induced hepatotoxicity.

Alcohol-induced liver disease (ALD) is a leading cause of non-accident-related deaths in the world. Identification of an early specific signature of ALD would aid in therapeutic intervention. Scoparone is an important constituent of Yinchenhao, and displayed bright prospects in hepatoprotective effect. However, its precise molecular mechanism has not been well explored. The present study was designed to assess the effects and possible mechanisms of scoparone against alcohol-induced liver injury. UPLC/ESI-Q-TOF/MS combined with pattern recognition approaches including PCA, and PLS-DA were integrated to get differentiating metabolites for the pathways and clarify mechanisms of disease, highlight insights into drug discovery. The results indicated four ions in the positive mode were characterized as potential differentiating metabolites which can be regulated by scoparone treatment, and suggested that therapeutic effect of scoparone could regulated the dysfunctions of citrate cycle, sphingolipid metabolism, taurine and hypotaurine.

Urinary ethyl glucuronide as a novel screening tool in patients pre- and post-liver transplantation improves detection of alcohol consumption.

UNLABELLED: Optimal selection of liver transplant candidates and early detection of alcohol relapse after orthotopic liver transplantation (OLT) is necessary to improve long-term outcomes. In this study, urinary ethyl glucuronide (uEtG) was prospectively evaluated as a novel screening tool for alcohol detection in the transplant setting. Overall, 141 liver transplant candidates and recipients, visiting the outpatient clinic for a total of 308 times, were included. At each visit, the alcohol markers, uEtG, ethanol, methanol, and carbohydrate-deficient transferrin (CDT), as well as the state markers, alanine transaminase, aspartate transaminase, gamma glutamyl transpeptidase (GGT), and mean corpuscular volume (MCV), were determined, then compared to patients' self-reports on alcohol intake. Urinary EtG significantly increased the detection rate of alcohol consumption, compared to the other alcohol markers (P < 0.001). In 93% of patients and at 92.5% of visits with positive alcohol markers, alcohol intake was detected by uEtG and/or CDT. Sensitivity and specificity of uEtG were 89.3% and 98.9% and of CDT were 25% and 98.6%, respectively. Urinary EtG was the best independent predictor of alcohol consumption in univariate and multivariate analysis (positive predictive value: 89.3%; negative predictive value: 98.9%; odds ratio: 761.1; P < 0.001). It showed a superior prediction rate, when compared to established alcohol and state markers, as well as to the combination of CDT with MCV and GGT, assessed by net reclassification improvement (NRI) (NRI: 1.01, P < 0.001; NRI: 1.755, P < 0.001). CONCLUSION: uEtG is a sensitive, specific, and reliable marker for the detection of recent alcohol intake pre- and post-OLT. In combination with CDT, uEtG should be considered as a tool for routine alcohol screening within the transplant setting.

UPLC-MS-based urine metabolomics reveals indole-3-lactic acid and phenyllactic acid as conserved biomarkers for alcohol-induced liver disease in the Ppara-null mouse model.

Since the development and prognosis of alcohol-induced liver disease (ALD) vary significantly with genetic background, identification of a genetic background-independent noninvasive ALD biomarker would significantly improve screening and diagnosis. This study explored the effect of genetic background on the ALD-associated urinary metabolome using the Ppara-null mouse model on two different backgrounds, C57BL/6 (B6) and 129/SvJ (129S), along with their wild-type counterparts. Reversed-phase gradient UPLC-ESI-QTOF-MS analysis revealed that urinary excretion of a number of metabolites, such as ethylsulfate, 4-hydroxyphenylacetic acid, 4-hydroxyphenylacetic acid sulfate, adipic acid, pimelic acid, xanthurenic acid, and taurine, were background-dependent. Elevation of ethyl-ß-d-glucuronide and N-acetylglycine was found to be a common signature of the metabolomic response to alcohol exposure in wild-type as well as in Ppara-null mice of both strains. However, increased excretion of indole-3-lactic acid and phenyllactic acid was found to be a conserved feature exclusively associated with the alcohol-treated Ppara-null mouse on both backgrounds that develop liver pathologies similar to the early stages of human ALD. These markers reflected the biochemical events associated with early stages of ALD pathogenesis. The results suggest that indole-3-lactic acid and phenyllactic acid are potential candidates for conserved and pathology-specific high-throughput noninvasive biomarkers for early stages of ALD.

Identification of noninvasive biomarkers for alcohol-induced liver disease using urinary metabolomics and the Ppara-null mouse.

Alcohol-induced liver disease (ALD) is a leading cause of nonaccident-related deaths in the United States. Although liver damage caused by ALD is reversible when discovered at the earlier stages, current risk assessment tools are relatively nonspecific. Identification of an early specific signature of ALD would aid in therapeutic intervention and recovery. In this study, the metabolic changes associated with ALD were examined using alcohol-fed male Ppara-null mouse as a model of ALD. Principal components analysis of the mass spectrometry-based urinary metabolic profile showed that alcohol-treated wild-type and Ppara-null mice could be distinguished from control animals without information on history of alcohol consumption. The urinary excretion of ethyl-sulfate, ethyl-beta-d-glucuronide, 4-hydroxyphenylacetic acid, and 4-hydroxyphenylacetic acid sulfate was elevated and that of the 2-hydroxyphenylacetic acid, adipic acid, and pimelic acid was depleted during alcohol treatment in both wild-type and the Ppara-null mice albeit to different extents. However, indole-3-lactic acid was exclusively elevated by alcohol exposure in Ppara-null mice. The elevation of indole-3-lactic acid is mechanistically related to the molecular events associated with development of ALD in alcohol-treated Ppara-null mice. This study demonstrated the ability of a metabolomics approach to identify early, noninvasive biomarkers of ALD pathogenesis in Ppara-null mouse model.

[Role of cholestasis in development of copper metabolism disorder in patients with chronic liver disease].

In this article was evaluated correlation between violations of copper exchange and the severity cholestasis at 162 of the patients with chronic liver diseases. There was high correlation between the level of AF and copper content in the blood. Also it was shown that patients with cholestasis significantly more frequently have observed hypercopperemy.

Metabolomic profiling of a modified alcohol liquid diet model for liver injury in the mouse uncovers new markers of disease.

Metabolomic evaluation of urine and liver was conducted to assess the biochemical changes that occur as a result of alcohol-induced liver injury. Male C57BL/6J mice were fed an isocaloric control- or alcohol-containing liquid diet with 35% of calories from corn oil, 18% protein and 47% carbohydrate/alcohol for up to 36 days ad libitum. Alcohol treatment was initiated at 7 g/kg/day and gradually reached a final dose of 21 g/kg/day. Urine samples were collected at 22, 30 and 36 days and, in additional treatment groups, liver and serum samples were harvested at 28 days. Steatohepatitis was induced in the alcohol-fed group since a 5-fold increase in serum alanine aminotransferase activity, a 6-fold increase in liver injury score (necrosis, inflammation and steatosis) and an increase in lipid peroxidation in liver were observed. Liver and urine samples were analyzed by nuclear magnetic resonance spectroscopy and electrospray infusion/Fourier transform ion cyclotron resonance-mass spectrometry. In livers of alcohol-treated mice the following changes were noted. Hypoxia and glycolysis were activated as evidenced by elevated levels of alanine and lactate. Tyrosine, which is required for l-DOPA and dopamine as well as thyroid hormones, was elevated possibly reflecting alterations of basal metabolism by alcohol. A 4-fold increase in the prostacyclin inhibitor 7,10,13,16-docosatetraenoic acid, a molecule important for regulation of platelet formation and blood clotting, may explain why chronic drinking causes serious bleeding problems. Metabolomic analysis of the urine revealed that alcohol treatment leads to decreased excretion of taurine, a metabolite of glutathione, and an increase in lactate, n-acetylglutamine and n-acetylglycine. Changes in the latter two metabolites suggest an inhibition of the kidney enzyme aminoacylase I and may be useful as markers for alcohol consumption.

Urinary ethyl glucuronide testing detects alcohol consumption in alcoholic liver disease patients awaiting liver transplantation.

This study compared measurement of urinary ethyl glucuronide (EtG), a conjugated minor ethanol metabolite with a longer detection window than ethanol itself, with breath alcohol testing and self-report as ways to disclose recent drinking by 18 liver transplant candidates with an alcoholic liver disease diagnosis that underwent an addiction group therapy program. At each therapy session, patients were questioned about any alcohol consumption in the intervening time, and they also performed a mandatory breath alcohol test, while observed urine samples for measurement of EtG were delivered on a voluntary basis. None of the patients ever admitted to intake of alcohol, and only 1 of 127 breath alcohol tests turned out positive. However, 9 patients showed positive EtG results in 24 (49%) of 49 urine samples. The individual frequency of urine samples being positive for EtG ranged from 22% to 100% with a mean value of 57%. Because 6 patients refused to provide urine on a total of 18 occasions, alcohol use might have been even more common. These results underscore the uncertainty of self-report data and the low sensitivity of breath alcohol testing as ways to disclose recent drinking, and underline the necessity of introducing sensitive and specific objective measures of recent alcohol consumption, such as EtG, in the transplantation setting.

The effect of rotenone on the urinary ethanol cycle in rats fed ethanol intragastrically.

Chronic ethanol ingestion alters mitochondrial function in the liver including inhibition of complex I of the electron transport chain. This leads to a shift in the NAD/NADH ratio to the reduced state when blood ethanol levels are high. Rotenone also inhibits complex I and induces a reduced state. The combination of ethanol feeding and rotenone toxicity should amplify the reduced state and block the cyclic increase and decrease in the rate of metabolism in the liver. The change in the redox state occurs during the urinary ethanol cycle in the intragastric tube feeding rat model of alcoholic liver disease. To test this hypothesis, rats were fed ethanol with rotenone and the 24-h urinary ethanol levels were measured daily. When ethanol was fed alone, the urinary ethanol cycle occurred. However, when ethanol was fed with rotenone the cycle was prevented and the urinary ethanol levels remained at the 200-mg% range. The rats fed ethanol or fed ethanol plus rotenone had the same increase in the pathology score and ALT elevations in the blood. Rotenone fed alone had the same normal values as the dextrose pair fed control rats. The results indicate that the UAL cycle is driven by fluctuation in the NAD/NADH ratio. When this fluctuation is blocked by rotenone, the cycle does not occur. It is concluded that the urinary ethanol cycle is dependent on cyclic fluctuation of the NAD/NADH ratio, which regulates the rate of ethanol elimination.

Reversibility of increased formation of catecholamines in patients with alcoholic liver disease.

BACKGROUND: While chronic alcohol abuse has been shown to be associated with increased production of catecholamines, little is known about the reversibility of this increased sympathetic activity and the influence of severity of alcoholic liver disease (ALD). The aim of the present study was to investigate whether the increase in urinary excretion rates and plasma levels of catecholamines in alcohol-abusing patients are reversible during prolonged abstinence, especially with respect to the severity of ALD. METHODS: Urinary excretion rates and plasma levels of noradrenaline (NA), adrenaline (A) and dopamine (DA) were determined in 15 subjects with mild to moderate ALD (ALD1) and in 7 alcoholic cirrhotics (ALD2) on admission and after 2 and 12 weeks of abstinence. Eight healthy males, age-matched to ALD1, served as controls (HC). RESULTS: Urinary excretion rates (24 h) and resting plasma concentrations of NA and A were increased in ALD1 and ALD2 about 2-fold, while those of DA were elevated only moderately compared to HC. During exercise under a load of 100 watts, the increases in plasma levels of NA and A with reference to the resting values were nearly identical in all three groups. Already after 2 weeks of abstinence, the urinary excretion rate of NA had nearly normalized in ALD1 but remained unchanged in ALD2. CONCLUSION: The marked enhancement of catecholamine production, especially that of NA, observed in actively drinking alcoholics is reversible under abstinence within a few weeks in subjects with mild to moderate ALD but only partially reversible in alcoholic cirrhosis.

Nutritional deficiencies in German middle-class male alcohol consumers: relation to dietary intake and severity of liver disease.

OBJECTIVE: The purpose of the present study was to compare the nutrient intake and the nutritional status between German middle-class alcohol consumers and non-drinkers. DESIGN: Cross-sectional study using patients with different stages of alcoholic liver disease (ALD) and healthy volunteers. SETTING: Southern Germany. SUBJECTS: Seventy-six hospitalized German middle-class alcohol consumers with different stages of alcoholic liver disease (ALD) and 22 healthy control subjects. METHODS: Subjects and controls were nutritionally assessed and mineral and vitamin content was measured in blood and urine. RESULTS: When compared with controls, alcohol consumers had significantly higher intakes of total calories, but intake of non-alcoholic calories did not differ between groups (P<0.05). Among drinkers, there was a decrease in percentage of energy derived from protein and fat and a significant increase in carbohydrates (P<0.05). With the exception of vitamin E, micronutrient intake of alcoholics was equal to that of controls; however, blood vitamin (vitamin C, retinol, lycopene, alpha- and gamma-carotene) and trace element (selenium, zinc) concentrations of alcohol-drinking patients were lower than those of non-drinkers. CONCLUSION: From the results of this study it is concluded that in German middle-class male alcohol consumers the status of several micronutrients is disturbed, although dietary intake hardly differs from that in non-alcoholic controls.