Patterns of IgA Autoantibody Generation, Inflammatory Responses and Extracellular Matrix Metabolism in Patients with Alcohol Use Disorder.

Recent data have emphasized the role of inflammation and intestinal immunoglobulin A (IgA) responses in the pathogenesis of alcoholic liver disease (ALD). In order to further explore such associations, we compared IgA titers against antigens targeted to ethanol metabolites and tissue transglutaminase with pro- and anti-inflammatory mediators of inflammation, markers of liver status, transferrin protein desialylation and extracellular matrix metabolism in alcohol-dependent patients with or without liver disease and in healthy controls. Serum IgAs against protein adducts with acetaldehyde (HbAch-IgA), the first metabolite of ethanol, and tissue transglutaminase (tTG-IgA), desialylated transferrin (CDT), pro- and anti-inflammatory cytokines, markers of liver status (GT, ALP) and extracellular matrix metabolism (PIIINP, PINP, hyaluronic acid, ICTP and CTx) were measured in alcohol-dependent patients with (n = 83) or without (n = 105) liver disease and 88 healthy controls representing either moderate drinkers or abstainers. In ALD patients, both tTG-IgA and HbAch-IgA titers were significantly higher than those in the alcoholics without liver disease (p < 0.0005 for tTG-IgA, p = 0.006 for Hb-Ach-IgA) or in healthy controls (p < 0.0005 for both comparisons). The HbAch-IgA levels in the alcoholics without liver disease also exceeded those found in healthy controls (p = 0.0008). In ROC analyses, anti-tTG-antibodies showed an excellent discriminative value in differentiating between ALD patients and healthy controls (AUC = 0.95, p < 0.0005). Significant correlations emerged between tTG-IgAs and HbAch-IgAs (rs = 0.462, p < 0.0005), CDT (rs = 0.413, p < 0.0001), GT (rs = 0.487, p < 0.0001), alkaline phosphatase (rs = 0.466, p < 0.0001), serum markers of fibrogenesis: PIIINP (rs = 0.634, p < 0.0001), hyaluronic acid (rs = 0.575, p < 0.0001), ICTP (rs = 0.482, p < 0.0001), pro-inflammatory cytokines IL-6 (rs = 0.581, p < 0.0001), IL-8 (rs = 0.535, p < 0.0001) and TNF-α (rs = 0.591, p < 0.0001), whereas significant inverse correlations were observed with serum TGF-ß (rs = -0.366, p < 0.0001) and CTx, a marker of collagen degradation (rs = -0.495, p < 0.0001). The data indicate that the induction of IgA immune responses toward ethanol metabolites and tissue transglutaminaseis a characteristic feature of patients with AUD and coincides with the activation of inflammation, extracellular matrix remodeling and the generation of aberrantly glycosylated proteins. These processes appear to work in concert in the sequence of events leading from heavy drinking to ALD.

Blood Cell Responses Following Heavy Alcohol Consumption Coincide with Changes in Acute Phase Reactants of Inflammation, Indices of Hemolysis and Immune Responses to Ethanol Metabolites.

Aberrations in blood cells are common among heavy alcohol drinkers. In order to shed further light on such responses, we compared blood cell status with markers of hemolysis, mediators of inflammation and immune responses to ethanol metabolites in alcohol-dependent patients at the time of admission for detoxification and after abstinence. Blood cell counts, indices of hemolysis (LDH, haptoglobin, bilirubin), calprotectin (a marker of neutrophil activation), suPAR, CD163, pro- and anti-inflammatory cytokines and autoantibodies against protein adducts with acetaldehyde, the first metabolite of ethanol, were measured from alcohol-dependent patients (73 men, 26 women, mean age 43.8 ± 10.4 years) at baseline and after 8 ± 1 days of abstinence. The assessments also included information on the quantities of alcohol drinking and assays for biomarkers of alcohol consumption (CDT), liver function (AST, ALT, ALP, GGT) and acute phase reactants of inflammation. At baseline, the patients showed elevated values of CDT and biomarkers of liver status, which decreased significantly during abstinence. A significant decrease also occurred in LDH, bilirubin, CD163 and IgA and IgM antibodies against acetaldehyde adducts, whereas a significant increase was noted in blood leukocytes, platelets, MCV and suPAR levels. The changes in blood leukocytes correlated with those in serum calprotectin (p < 0.001), haptoglobin (p < 0.001), IL-6 (p < 0.02) and suPAR (p < 0.02). The changes in MCV correlated with those in LDH (p < 0.02), MCH (p < 0.01), bilirubin (p < 0.001) and anti-adduct IgG (p < 0.01). The data indicates that ethanol-induced changes in blood leukocytes are related with acute phase reactants of inflammation and release of neutrophil calprotectin. The studies also highlight the role of hemolysis and immune responses to ethanol metabolites underlying erythrocyte abnormalities in alcohol abusers.

Assessment of alcohol consumption in depression follow-up using self-reports and blood measures including inflammatory biomarkers.

AIMS: Alcohol consumption has been suggested a major role in the pathogenesis and prognosis of depression. However, reliable identification of hazardous drinking continues to be problematic. We compared the accuracy of different biomarkers and self-reports of alcohol consumption in the follow-up study of depression. METHODS: Data from 202 patients with major depressive disorder were obtained through self-reports, AUDIT and AUDIT-C questionnaires and biomarker analyses. The clinical assessments and measurements of biomarkers (GT, CDT, GT-CDT-combination, MCV, ALT, AST, hs-CRP, IL-6) were performed at baseline and after six months of treatment. Based on self-reported alcohol intake at baseline the patients were classified to three subgroups. RESULTS: About 27.2% of patients were categorized to high-risk drinkers, 26.3% low-risk drinkers and 46.5% abstainers. High-risk drinkers showed significantly higher mean values of GT, CDT, GT-CDT-combination and IL-6 than abstainers, diagnostic accuracy being highest with the combined marker of GT-CDT. The accuracy of AUDIT and AUDIT-C to detect high-risk drinking was also significant. During follow-up, the differences observed in the biomarkers at baseline disappeared together with recovery from depression. CONCLUSIONS: Our data suggest the combined use of GT-CDT and AUDIT questionnaires to improve the identification of drinking of patients with depression. This approach could be useful for improving treatment adherence and outcome in depressed patients.

Serum soluble urokinase plasminogen activator receptor in alcoholics: relation to liver disease severity, fibrogenesis, and alcohol use.

BACKGROUND AND AIM: Heavy alcohol consumption may lead to development of liver disease and the need for non-invasive parameters for detecting those at risk is widely acknowledged. METHODS: We measured serum soluble urokinase-type plasminogen activator receptor (suPAR) levels from 63 patients with alcoholic liver disease (ALD), 57 heavy drinkers without apparent liver disease, and 39 controls who were either moderate drinkers or abstainers. RESULTS: The highest serum suPAR concentrations were detected in patients with ALD (P < 0.001) showing high diagnostic accuracy in differentiating ALD patients from heavy drinkers without liver disease (area under curve 0.921, P < 0.001). Levels of suPAR correlated positively with serum markers of fibrogenesis (aminoterminal propeptide of type III procollagen and hyaluronic acid) (P < 0.001), with clinical (combined clinical and laboratory index P < 0.01) and morphological (combined morphological index P < 0.05) indices of liver disease severity and with the stage of fibrosis (P < 0.01). The suPAR concentrations were also elevated in heavy drinkers when compared with healthy controls (P < 0.001). CONCLUSION: The data indicate that serum suPAR concentrations are increased as a result of heavy alcohol consumption and further with development of ALD, showing a good diagnostic performance in detecting those with liver disease. The association with the histological severity of ALD and correlation with fibrosis indicates potential of serum suPAR also as a prognostic marker in ALD.

Comparison of ethyl glucuronide and carbohydrate-deficient transferrin in different body fluids for post-mortem identification of alcohol use.

AIMS: Alcohol abuse is a major risk factor for premature death. Confirming the role of alcohol consumption in cause-of-death investigations has, however, remained difficult, due to lack of reliable biomarkers. METHODS: We compared ethyl glucuronide (EtG) and carbohydrate-deficient transferrin (CDT) assays from serum, urine, cerebrospinal fluid and vitreous humor in a forensic autopsy population with either a positive (n = 38) or negative (n = 22) history of alcohol abuse based on detailed medical and police records and forensic toxicological investigations. RESULTS: A positive blood alcohol concentration (median 1.15‰, range 0-3.3‰) was found in 26/38 (68%) of the cases with a documented history of alcohol abuse. EtG concentrations (mean ± SD) in urine (339 ± 389 mg/l, P < 0.001), vitreous humor (4.2 ± 4.8 mg/l, P < 0.001), serum (6.9 ± 8.9 mg/l, P < 0.01) and cerebrospinal fluid (1.7 ± 2.7 mg/l, P < 0.01) were significantly higher among the cases with a positive history of alcohol use than those in the alcohol-history negative group, whereas in corresponding comparisons CDT was significantly different only in cerebrospinal fluid (4.3 ± 2.1 vs. 2.3 ± 0.6%, P < 0.05). The highest sensitivities (92%) in detecting ante-mortem alcohol use were obtained for urine and vitreous humor EtG assays. CONCLUSION: Our data indicate that measurements of EtG in urine or vitreous humor show the highest diagnostic accuracies in post-mortem investigations of excessive alcohol consumption and can be recommended for routine applications.

First trimester biochemistry at different maternal ages.

BACKGROUND: The performance of first trimester biochemical screening was compared at different pregnancy weeks and maternal ages during 2002-2008 in a screened population of 76,949 women. METHODS: The detection rates, as well as the median multiples of a median (MOMs) of free ß-human chorionic gonadotropin (free ß-hCG) and pregnancy-associated plasma protein-A (PAPP-A), were compared between completed gestational weeks 8-13 and between different maternal ages separated into 5-year groupings. RESULTS: The number of singleton Down syndrome pregnancies was 221. The median age of the screened women was 30 years and the proportion of women aged ≥ 35 years 16.9%. The median age of the women with a Down syndrome pregnancy was 37 years. In women aged <35 years, the biochemical markers provided a detection rate of only 38.6%, whereas in women aged ≥ 35 years, the biochemical markers detected 82.7% of cases (p<0.01). CONCLUSIONS: Biochemical screening works best amongst women aged ≥ 35 years. For younger mothers aged <35 years, combined screening should be the method of choice.

More invasive procedures are done to detect each case of Down's syndrome in younger women.

OBJECTIVE: To evaluate the performance of first-trimester combined screening in 5-year periods according to maternal age in a low-risk population. DESIGN: A prospective study. SETTING: Multicenter study in Finland. POPULATION: A total of 76949 voluntary women with singleton pregnancies participated in first-trimester combined screening in public healthcare between 1 May 2002 and 31 December 2008. METHODS: The serum samples were analyzed using the PerkinElmer AutoDELFIA® time-resolved fluoroimmunoassay kit for the measurement of pregnancy-associated plasma protein-A and free beta-human chorionic gonadotropin. Nuchal translucency was measured by trained personnel (midwives or physicians) in a university or central hospital. MAIN OUTCOME MEASURES: Performance, detection rate, false positive rate and the number of invasive procedures needed to detect a single case of Down's syndrome were analyzed. RESULTS: There was a direct connection between maternal age and the prevalence of Down's syndrome with a low prevalence in young women being 1:1 193 in the 25-29 age group and 1:150 in the 35-39 age group. Consequently, for a fixed false positive rate of 5%, the number of invasive procedures needed to detect one case of Down's syndrome is higher in younger women to achieve the same detection rate. CONCLUSIONS: In combined first trimester screening the risk for Down's syndrome is individual, varying with maternal age. This should be taken into consideration when counseling women.

Comparison of serum calprotectin, a marker of neutrophil activation, and other mediators of inflammation in response to alcohol consumption.

AIMS: Previous studies have indicated that heavy alcohol intake stimulates inflammation and impairs the body's ability to regulate inflammation. The aim of this study was to compare changes in neutrophil calprotectin and a wide spectrum of other inflammatory mediators in response to heavy alcohol drinking. METHODS: Serum calprotectin (a marker of neutrophil activation), suPAR, CD163, and pro- (IL-6, IL-8, TNF-α) and anti-inflammatory (IL-10, TGF-ß) cytokines were measured from 61 alcohol-dependent subjects (46 men, 15 women, mean age 43.6 ± 11.0 years) at the time of admission for detoxification and after 8 ± 2 days of abstinence. These biomarkers were also measured from age- and sex-matched healthy controls representing abstainers or light drinkers. The clinical assessments included detailed clinical interviews on the amounts and patterns of alcohol consumption and assays for biomarkers of alcohol consumption (GGT, CDT, MCV, GGT-CDT) and liver function (AST, ALT). RESULTS: The subjects with alcohol use disorder showed significantly higher concentrations of serum calprotectin (p < 0.0005), suPAR (p < 0.01), CD163 (p < 0.01), IL-6 (p < 0.0005), IL-8 (p < 0.0005), TNF-α (p < 0.001), and IL-10 (p < 0.0005) than healthy controls. These inflammatory mediators, except for CD163, remained elevated after the 8 ± 2-day period of supervised abstinence, which resulted in significant decreases in the biomarkers of alcohol consumption and indices of liver status. The AUC (0.855) for serum calprotectin in differentiating between the heavy drinkers and healthy controls was equal or equivalent with those of the conventional biomarkers of alcohol consumption (GGT:0.835 or CDT:0.803). CONCLUSIONS: The data indicate that neutrophil calprotectin is released in response to heavy alcohol intake in a sensitive manner and may be associated with perpetuation of inflammation in patients with alcohol use disorder. Serum calprotectin may also prove to be a useful biomarker for inflammatory activity in alcohol-consuming patients.

Evaluation of reference intervals for biomarkers sensitive to alcohol consumption, excess body weight and oxidative stress.

BACKGROUND: Unexplained liver enzyme activities are often found in health screening programs and constitute an increasingly common cause for referral to specialized clinics. Recent studies have indicated that both excess body weight and alcohol consumption may lead to metabolic aberrations which are readily reflected in the activities of liver enzymes in circulation. MATERIALS AND METHODS: We compared various laboratory markers and their upper normal limits in relation to information on alcohol consumption and BMI in a large population of apparently healthy individuals collected from Nordic countries. RESULTS: Based on the data obtained from normal weight abstainers (BMI 19-25 kg/m(2)) the upper normal limits in men should be 50 U/L for ALT, and 45 U/L (<40 years) and 70 U/L (>or=40 years) for GGT, while the current recommendations are 70 U/L, 80 U/L, and 115 U/L, respectively. Already in comparisons between normal weight abstainers and corresponding moderate drinkers notable impacts (+14% - +74%) on upper limits for these analytes were seen, which further grew when adiposity occurred together with alcohol drinking (+75% - +186%, BMI >or=27 kg/m(2)). In addition to liver enzymes, similar changes were also found for uric acid. CONCLUSIONS: Alcohol consumption and excess body weight even in apparently healthy individuals have a significant influence on liver enzyme activities, which may be due to a cumulative oxidative stress burden. The metabolic changes induced by adiposity or ethanol intake should be considered in the definition of normal ranges for all laboratory parameters sensitive to oxidative stress.

Status of inflammation and alcohol use in a 6-month follow-up study of patients with major depressive disorder.

INTRODUCTION: The studies on the relationship between alcohol consumption, the status of inflammation, and depression have produced conflicting results. In this study, we followed patients with major depressive disorders by monitoring biomarkers of inflammation together with biomarkers of heavy alcohol use. METHOD: The levels of IL-6 (interleukin-6), IL-8 (interleukin-8), hs-CRP (high sensitivity C-reactive protein), YKL-40 (also known as Chitinase-3-like protein 1 or CHI3L1), and biomarkers of alcohol consumption and liver status (GT, CDT, ALT, alkaline phosphatase) were measured at baseline and after 6 months of psychiatric treatment from 242 patients suffering from current major depressive disorder (MDD) with (n = 99) or without (n = 143) alcohol use disorder (AUD). RESULTS: At baseline, the patients with MDD + AUD showed higher levels of inflammatory biomarkers IL-6 (p < 0.001), hs-CRP (p < 0.01), YKL-40 (p < 0.05), and biomarkers of alcohol consumption, than the corresponding group of non-AUD patients. These differences disappeared during follow-up and recovery from depression. The level of IL-8 decreased significantly in both AUD (p < 0.05) and non-AUD (p < 0.05) patients. During follow-up, the biomarkers of alcohol consumption, GT and CDT, in AUD patients were found to decrease in parallel with serum YKL-40 levels. CONCLUSIONS: Alcohol consumption appears to modulate the status of inflammation in depressive patients. A more systematic use of biomarkers of inflammation together with biomarkers of alcohol consumption and liver status may prove to be of value in a more comprehensive assessment and treatment of patients with depression.

Immunoassay for ethyl glucuronide in vitreous humor: a new tool for postmortem diagnostics of alcohol use.

Although excessive alcohol consumption plays a major role in fatal events, the role of alcohol use as a possible contributing factor at the time of death is not easy to establish due to lack of suitable biomarkers for postmortem analyses. We used an immunological approach to measure ethyl glucuronide (EtG) concentrations from vitreous humor (VH) and serum from 58 individuals representing a forensic autopsy population of cases with either a well-documented history of excessive alcohol use (n=37) or cases without such history (n=21), according to medical and police records and blood alcohol determinations (BAC). The immunoassay was based on the Microgenics DRI-EtG EIA reagents applied on an automated Abbott Architect c8000 clinical chemistry analyzer. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) determination of EtG and ethyl sulfate (EtS) was used as a reference method. At a cut-off of 0.3mg/l for VH-EtG, the immunoassay correctly identified 92% of the cases with a history of excessive alcohol use, whereas the BAC was positive (cut-off 10mg/dl) in 68% of the cases. A significant correlation emerged between VH-EtG and serum EtG (r=0.77, p<0.001) and between VH-EtG and BAC (r=0.62, p<0.001), although VH-EtG was frequently elevated also in cases with no detectable BAC. The EtG immunoassay showed a strong correlation with the LC-MS/MS reference method (r=0.94, p<0.001) and there was 100% agreement in the frequency of marker positive and negative findings between the immunoassay EtG results and the LC-MS/MS analysis of EtG and EtS. The present data indicate that the immunoassay for VH-EtG is a useful forensic tool for screening of antemortem alcohol use.