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.

Gender-dependent impacts of body mass index and moderate alcohol consumption on serum uric acid--an index of oxidant stress status?

Uric acid seems to be causally involved in a variety of medical disorders involving oxidative stress. Although alcohol abuse and obesity are known to increase serum uric acid, the interactions between moderate drinking, adiposity, and uric acid metabolism have remained poorly understood. We examined serum uric acid concentrations from 2062 apparently healthy volunteers (970 men, 1092 women) reporting either no alcohol (abstainers) or <40 g of ethanol consumption per day (moderate drinkers). The study population was further classified according to BMI as follows: <19 (underweight), 19-25 (normal weight), 25-30 (overweight), and >30 (obese). Serum uric acid concentrations in male moderate drinkers were significantly higher, and in females they were lower, than in the corresponding groups of abstainers. In the BMI-based subgroups, the highest concentrations were found in those who were overweight or obese. Significant two-factor interactions occurred between gender and drinking status (p<0.001) and between gender and BMI (p<0.02). Serum uric acid also correlated with indices of hepatocellular health (GGT, ALT, AST). The data indicate distinct gender-dependent impacts of alcohol consumption and BMI on serum uric acid. These findings should be applicable to the assessment of oxidative stress status and associated morbidity in alcohol consumers and individuals with excess body weight.

Biomarkers of liver status in heavy drinkers, moderate drinkers and abstainers.

AIMS: Although a wide variety of biomarkers reflecting liver status are known to be influenced by excessive ethanol consumption, the dose-response relationships between ethanol intake and marker changes have remained less understood. METHODS: Serum gamma-glutamyltransferase (GGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT) activities, and ferritin and albumin protein concentrations were compared in a large population of heavy drinkers (105 men, 28 women), moderate drinkers (781 men, 723 women) and abstainers (252 men, 433 women), who were devoid of apparent liver disease. RESULTS: In heavy drinkers, serum GGT, AST, ALT, ferritin and albumin were all significantly higher than in moderate drinkers or abstainers (P < 0.001 for all comparisons). The highest incidences of elevated values were found for GGT (62%) followed by AST (53%), ALT (39%), ferritin (34%) and albumin (20%). Serum GGT (P < 0.001), ALT (P < 0.01) and ferritin (P < 0.05) in moderate drinkers were also higher than the levels observed in abstainers. When the study population was further divided into subgroups according to gender, significant differences between moderate drinkers and abstainers in GGT and ALT were noted in men whereas not in women. CONCLUSIONS: The data demonstrate that biomarkers of alcohol abuse and liver function may respond to even rather low levels of ethanol intake in a gender-dependent manner, which should be implicated in studies on the early-phase interactions of ethanol and the liver and in the definition of normal ranges for such biomarkers.

Effect of moderate alcohol consumption on liver enzymes increases with increasing body mass index.

BACKGROUND: Although both ethanol consumption and overweight alter the activities of hepatic enzymes in circulation, the differentiation of an alcohol or nonalcohol basis for such changes remains problematic. The magnitude of alterations occurring among moderate drinkers has remained obscure. OBJECTIVE: We examined the links between moderate ethanol consumption, body mass index (BMI; in kg/m(2)), and liver enzymes. DESIGN: Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyltransferase (GGT) were recorded from 2,164 apparently healthy participants (1,028 men, 1,136 women) reporting either no alcohol (abstainers) or <40 g ethanol consumption per day (moderate drinkers). The study population was further classified according to BMI as follows: <19 (underweight), > or =19 and <25 (normal weight), > or =25 and <30 (overweight), and > or =30 (obese). RESULTS: Serum ALT (P < 0.05) and GGT (P < 0.001) but not AST (P = 0.805) activities in moderate drinkers were higher than those in abstainers. For all enzymes, a significant main effect was observed of increasing BMI, which was more striking in moderate drinkers than in abstainers. Tests of between-subjects effects indicated significant interactions with sex and drinking status, although not with sex and BMI. CONCLUSIONS: The effect of moderate alcohol consumption on liver enzymes increases with increasing BMI. These findings should be considered in the clinical assessment of overweight alcohol consumers and in the definition of normal ranges for liver enzymes. These results may also help to develop new approaches for examining patients with fatty liver induced by either ethanol or adiposity.

Demonstration of ethanol-induced protein adducts in oral leukoplakia (pre-cancer) and cancer.

BACKGROUND: Excessive alcohol consumption is a common cause for upper gastrointestinal tract cancers. However, the primary mechanisms of alcohol-induced carcinogenesis have remained poorly defined. METHOD: We examined the generation and subcellular distribution of protein adducts with acetaldehyde (AA), the first metabolite of ethanol, and end products of lipid peroxidation, malondialdehyde (MDA) and 4-hydroxynonenal (HNE), from oral biopsy specimens obtained from 36 subjects (11 British, 25 Japanese) reporting alcohol misuse. All patients had been diagnosed with oral pre-cancer (leukoplakia, n = 7) or squamous cell carcinoma (SCC; n = 29). Automated immunostaining for AA, MDA and HNE adducts was performed using monospecific antibodies. RESULTS: Positive staining for AA, MDA and HNE adducts was observed in the dysplastic or malignant epithelial cells, HNE being relatively the most abundant adduct species. The subgroup of Japanese patients had higher levels of AA and MDA, although not HNE, than the British sample. When the material was divided to those with SCC or leukoplakia, MDA adducts but not the other antigens were more prominent in the former group. Significant correlations were found between the different adducts (AA vs. MDA, r = 0.68, P < 0.001; AA vs. HNE, r = 0.47, P < 0.01 and MDA vs. HNE, r = 0.59, P < 0.001). In addition, cytochrome P450 2E1 staining was found in these samples, correlating with both AA and MDA adducts. CONCLUSION: The data indicates that AA- and lipid peroxidation-derived adducts are formed in oral tissues of alcohol misusers with oral leukoplakia and cancer. The findings also support a pathogenic role of AA and excessive oxidative stress in carcinogenesis.

Co-occurrence of IgA antibodies against ethanol metabolites and tissue transglutaminase in alcohol consumers: correlation with proinflammatory cytokines and markers of fibrogenesis.

IgA antibodies to tissue transglutaminase have been suggested to be specific indicators of celiac disease. However, no studies have addressed the relationships between such antibodies and alcohol abuse, which is also a common cause of IgA-isotype immune responses and tissue injury in the gastrointestinal tract and liver. Here, measurements of specific IgAs against tissue transglutaminase and proteins modified by acetaldehyde, the first metabolite of ethanol, showed significantly higher levels of both antibodies in alcoholic liver disease patients than in healthy controls or heavy drinkers without liver disease. These antibodies also significantly co-occurred in heavy drinkers without liver disease, moderate drinkers, and abstainers, and correlated with biomarkers of alcohol consumption, proinflammatory cytokines and markers of fibrogenesis. The data suggests a link between such immune responses, perturbations in cytokine profiles and fibrogenesis, which should be implicated in studies on the pathogenesis and diagnosis of ethanol-induced tissue injury and celiac disease.

The cytoprotective effect of alpha-tocopherol and daidzein against d-galactosamine-induced oxidative damage in the rat liver.

We hypothesized that the hepatotoxicity that develops after the induction of oxidative stress (induced by d-galactosamine [GalN]) can be ameliorated by alpha-tocopherol (ATC) and the soy isoflavone daidzein. To test this, we ranked and assigned male Wistar rats into 6 groups, which involved pretreatment (ATC or daidzein) for 1 hour followed by treatment (GalN) for 23 hours. Histopathologic analysis showed that GalN administration induced marked necrosis (P < .001), steatosis (P < .001), both lobular and portal inflammations (P < .001), overall histopathologic score (P < .001), and activation of caspase-3 in the liver (P < .001). Immunohistochemical staining of malondialdehyde-protein adducts, a measure of oxidative stress, was increased in response to GalN (P < .001). Paradoxically, there were increases in total (P < .05) and cytosolic superoxide dismutase (P < .001) activities after GalN administration, indicative of an up-regulation of antioxidant defenses. The concentration of total protein (P < .001), albumin (P < .01), and globulin fractions (P < .001) in the plasma, as well as the activity of aspartate aminotransferase (P < .001), was significantly perturbed after GalN treatment, reflective of overall acute hepatic injury. Administration of daidzein showed a significant amelioration of the Ga1N-induced increase in malondialdehyde-protein adducts (P < .01) and cytosolic superoxide dismutase activities (P < .01) in the liver. However, all other variables were not significantly altered in response to daidzein. In response to ATC pretreatment, the total histopathologic score (P < .05), degree of necrosis (P < .05), and both lobular (P < .05) and portal (P = .05) inflammations were significantly ameliorated. To conclude, both daidzein and ATC protect the liver against oxidative damage possibly via different pathways.

An inverse relationship between markers of fibrogenesis and collagen degradation in patients with or without alcoholic liver disease.

OBJECTIVES: Excessive deposition of collagen leading to cirrhosis is a major complication of alcohol abuse. However, the mechanisms behind the accumulation of the extracellular matrix proteins are poorly understood. METHODS: We measured serum markers of collagen degradation (beta-CTx), fibrogenesis (PINP, PIIINP), and pro- and anti-inflammatory cytokines from 84 male heavy drinkers, who were either with (N = 52) or without (N = 32) clinical or histological signs of alcoholic liver disease (ALD), and from 20 healthy nonalcoholic controls. RESULTS: Serum beta-CTx levels in ALD patients were significantly lower than in healthy controls or in the alcoholics without liver disease, while PINP and PIIINP, reflecting type I and type III collagen synthesis, respectively, were significantly increased. The alcoholics without liver disease showed values, that were not significantly different from those of healthy controls. Serum beta-CTx correlated negatively with serum PIIINP and proinflammatory cytokines (IL-2, IL-6, IL-8, TNF-alpha), and positively with anti-inflammatory cytokines (IL-1, TGF-beta), whereas serum PIIINP correlated positively with these proinflammatory cytokines and negatively with the anti-inflammatory cytokines. Calculation of PIIINP/beta-CTx ratio was found to yield an excellent sensitivity (94%) and specificity (98%) in differentiating the alcoholics with liver disease. CONCLUSION: The present findings indicate a positive relationship between markers of collagen biosynthesis and proinflammatory cytokines, and a negative relationship between these markers and a marker of collagen degradation and anti-inflammatory cytokines, suggesting that a disturbed balance in these cellular responses may facilitate fibrogenesis and play a pivotal role in the pathogenesis of ALD. These findings should also be implicated in the development of noninvasive tools for discriminating individuals at risk for fibrogenesis.

IgAs against acetaldehyde-modified red cell protein as a marker of ethanol consumption in male alcoholic subjects, moderate drinkers, and abstainers.

BACKGROUND: Alcohol abuse has been shown to result in the production of antibodies against acetaldehyde-modified epitopes in proteins. However, as yet, only limited information has been available on the clinical usefulness of such responses as markers of hazardous drinking. METHODS: We developed an ELISA to measure specific IgAs against acetaldehyde-protein adducts. This method was evaluated in cross-sectional and follow-up studies on male heavy drinkers with a current ethanol consumption of 40 to 540 g/d (n=40), moderate drinkers consuming 1 to 40 g/d (n=25), and abstainers (n=16). The clinical assessments included detailed interviews on the amounts and patterns of ethanol consumption and various biochemical markers of alcohol abuse and liver function. RESULTS: The mean antiadduct IgAs (198+/-28 U/L) in the alcohol abusers were significantly higher than those in the moderate drinkers (58+/-11 U/L, p<0.001) or abstainers (28+/-8 U/L, p<0.001). The values of moderate drinkers were also higher than those in abstainers (p<0.05). The amount of ethanol consumed during the period of 1 month preceding blood sampling correlated strongly with antiadduct IgAs (r=0.67, p<0.001). The sensitivity (73%) and specificity (94%) of this marker were found to exceed those of the conventional laboratory markers of alcohol abuse in comparisons contrasting heavy drinkers with abstainers although not in comparisons contrasting heavy drinkers with moderate drinkers. During abstinence, antiadduct IgAs disappeared with a mean rate of 3% per day. In additional analyses of possible marker combinations, antiadduct IgAs, together with CDT, were found to provide the highest sensitivity and specificity. CONCLUSIONS: Measurements of antiadduct IgAs may provide a new clinically useful marker of alcohol abuse, providing a close relationship between marker levels and the actual amounts of recent ethanol ingestion.

Age-related changes on serum ggt activity and the assessment of ethanol intake.

AIMS: Gamma-glutamyl transferase (GGT) is a commonly used marker of ethanol abuse. However, although increasing age has also been suggested to elevate serum GGT activities, the magnitude of such effects on GGT in the assessment of ethanol intake have remained poorly defined. METHODS: GGT activities from 208 heavy drinkers were compared with those from a reference population including 1330 moderate drinkers and 1160 abstainers, who were further classified to following age intervals: 18-30, 30-50, 50-70, and >70 years. RESULTS: GGT activities increased with increasing age until after 70 years decreasing values were noted in male abstainers. The heavy drinkers belonging to age groups 18-30, 30-50, and 50-70 years showed 2.7-, 8.0-, and 6.9-fold higher mean GGT activities than those in the corresponding groups of abstainers, respectively. The values in the group of moderate drinkers also exceeded those of abstainers in all age groups of men, whereas in women the difference was significant only among those aged 18-30 years. CONCLUSIONS: The data indicate that GGT activities respond to ethanol intake in an age-dependent manner, which should be considered in the clinical use of GGT measurements for detecting alcohol use disorders.

Additive effects of moderate drinking and obesity on serum gamma-glutamyl transferase activity.

BACKGROUND: Gamma-glutamyl transferase (GGT) is a widely used index of liver induction and a marker of alcohol overconsumption. Obesity has also been suggested to elevate serum GGT activities. OBJECTIVE: The aim was to examine the links between moderate ethanol consumption, obesity, and GGT activities. DESIGN: GGT values were recorded from 2490 persons (1184 men and 1306 women) who reported either no alcohol use (abstainers) or 1-40 g ethanol consumption per day (moderate drinkers). The study population was additionally classified according to body mass index (BMI; in kg/m2) as follows: < 19 (underweight), > or = 19 and < 25 (normal weight), > or = 25 and < 30 (overweight), and 30 (obese). RESULTS: Significant main effects of sex (P < 0.0001), drinking habits (P < 0.01), and BMI (P < 0.001) on serum GGT activities were observed. The values were higher in the men than in the women and higher in those with higher BMIs. The highest activities were found to occur in persons with moderate drinking combined with overweight or obesity. A significant positive correlation between GGT and BMI (P < 0.0001) was observed, which was stronger for the men (r = 0.24) than for the women (r = 0.15, P < 0.05 for the difference between correlations). CONCLUSION: The data indicate that serum GGT activities may respond to moderate drinking and overweight in an additive manner; this should be considered in the clinical use of GGT measurements and when defining normal GGT values in health care.

Comparison of the combined marker GGT-CDT and the conventional laboratory markers of alcohol abuse in heavy drinkers, moderate drinkers and abstainers.

AIMS: A combined index based on gamma-glutamyltransferase (GGT) and carbohydrate-deficient transferrin (CDT) measurements (GGT-CDT) has been recently suggested to improve the detection of excessive ethanol consumption. The aim of this work was to compare GGT-CDT with the conventional markers of alcohol abuse in individuals with a wide variety of alcohol consumption. METHODS: A cross-sectional and follow-up analysis was conducted in a sample of 165 heavy drinkers, consuming 40-540 g of ethanol per day, and 86 reference individuals who were either moderate drinkers (n = 51) or abstainers (n = 35). RESULTS: GGT-CDT (5.35 +/- 1.08) in the heavy drinkers was significantly higher than in the reference individuals (3.30 +/- 0.37). The sensitivity of GGT-CDT (90%) in correctly classifying heavy drinkers exceeded that of CDT (63%), GGT (58%), mean corpuscular volume (MCV) (45%), aspartate aminotransferase (AST) (47%), and alanine aminotransferase (ALT) (50%), being also essentially similar for alcoholics with (93%) or without (88%) liver disease. When comparing the data using either moderate drinkers or abstainers as reference population, the sensitivity of GGT-CDT, CDT, and ALT remained unchanged whereas the sensitivity of GGT, MCV, and AST was found to show variation. CONCLUSIONS: GGT-CDT improves the sensitivity of detecting excessive ethanol consumption as compared with the traditional markers of ethanol consumption. These findings should be considered in the assessment of patients with alcohol use disorders.

Long-term ethanol consumption and macrocytosis: diagnostic and pathogenic implications.

Although excessive alcohol consumption is known to elevate the mean cell volume (MCV) of erythrocytes, the relationships among the intensity of ethanol exposure, the generation of abnormal red blood cell indices, and the underlying pathogenic mechanisms have remained unclear. The authors examined 105 alcoholics with a wide range of ethanol consumption (40-500 g of ethanol/day), 62 moderate drinkers (mean consumption 1-40 g/day), and 24 abstainers, who underwent detailed interviews, measurements of blood cell counts, markers of liver status, and circulating antibodies against ethanol-derived protein modifications. Follow-up information was collected from healthy volunteers with detailed records on drinking habits. Data from the NORIP project for laboratory parameters in apparently healthy moderate drinkers or abstainers (n = 845) were used for reference interval comparisons. The highest MCV (P < 0.001) and mean cell hemoglobin (MCH) (P < 0.01) occurred in the alcoholics. However, the values in the moderate drinkers also responded to ethanol intake such that the upper normal limit for MCV based on the data from moderate drinkers was 98 fl, as compared with 96 fl from abstainers. Follow-up cases with carefully registered drinking habits showed parallel changes in MCV and ethanol intake. Anti-adduct IgA and IgM against acetaldehyde-induced protein modifications were elevated in 94% and 64% of patients with high MCV, respectively, the former being significantly less frequent in the alcoholics with normal MCV (63%) (P < 0.05). The data indicate dose-related responses in red blood indices upon chronic ethanol consumption, which may also be reflected in reference intervals for hematological parameters in health care. Generation of immune responses against acetaldehyde-modified erythrocyte proteins may be associated with the appearance of such abnormalities.

Serum gamma-glutamyl transferase in alcoholics, moderate drinkers and abstainers: effect on gt reference intervals at population level.

AIMS: To clarify in the association between amount of ethanol consumption and serum gamma-glutamyl transferase (GT) levels. METHODS: GT values were measured from 195 individuals with a wide variety of well-documented ethanol consumption assessed by detailed personal interviews using a time-line follow-back technique. These included 103 heavy drinkers (90 men, 13 women) and 92 healthy volunteers (54 men, 38 women) who were either abstainers (n = 30) or moderate drinkers (n = 62). For comparisons, data were collected from GT measurements for establishing GT reference intervals from 2485 healthy volunteers including 1156 abstainers and 1329 moderate drinkers. RESULTS: GT values in the individuals whose mean ethanol consumption exceeded 40 g of ethanol per day were significantly higher than those in the moderate drinkers with a mean consumption of 1-40 g/day (P < 0.001) or in abstainers (P < 0.001). The GT values in the group of moderate drinkers also exceeded those of the abstainers (P < 0.001). The upper normal GT limits obtained from the data from abstainers were markedly lower (men 45 U/l, women 35 U/l) than those obtained from the population of moderate drinkers (men 66 U/l, women 40 U/l). CONCLUSIONS: Serum GT concentrations may respond to relatively low levels of ethanol consumption, which should be considered when defining GT reference intervals. The continuous increase in alcohol consumption at population level may lead to increased GT cut-off limits and hamper the detection of alcohol problems and liver affection in their early phase.

Immune Responses to Ethanol Metabolites and Cytokine Profiles Differentiate Alcoholics with or without Liver Disease.

OBJECTIVES: Excessive alcohol consumption is associated with the generation of antibodies against neoantigens induced by ethanol metabolism. However, the associations between such immune responses, ethanol consumption, and liver injury remain unclear. METHODS: Eight-six male alcoholics with (n=54) or without (n=32) liver disease, and 20 male volunteers (6 abstainers, 14 moderate drinkers) underwent clinical, morphological, and biochemical assessments of liver status and ethanol consumption. RESULTS: Antiacetaldehyde adduct IgAs in both groups of alcoholics were significantly higher than those in the controls. Elevated IgGs occurred in patients with liver disease, whereas IgMs were high in the heavy drinkers without apparent liver disease. Liver disease patients had high levels of both proinflammatory (IL-2, IL-6, IL-8, TNF-alpha) and antiinflammatory (IL-10) cytokines, whereas those without liver disease showed elevated IL-6, IL-8, and IL-10 only. Ethanol consumption correlated significantly with antiadduct IgA and IL-6 levels, which also showed parallel changes upon abstinence. CONCLUSIONS: Alcoholic liver disease is associated with the generation of IgAs and IgGs against acetaldehyde-derived antigens and enhanced levels of both pro- and antiinflammatory cytokines, whereas elevated IgA, IL-6, and IL-10 characterize alcoholics without liver disease. These data suggest that immunological mechanisms may play a role in the sequence of events leading to liver disease in some patients with excessive drinking.

Cultures of human embryonic stem cells: serum replacement medium or serum-containing media and the effect of basic fibroblast growth factor.

Human embryonic stem (hES) cells have traditionally been cultured in medium containing fetal calf serum (FCS) and mouse fibroblasts as feeder cells. The use of animal derived materials carries a risk of transmitting animal pathogens, and they are not optimal in cultures aimed at cell transplantation in humans. This technical study aiming at facilitating IVF units to establish new hES cell lines, has systematically compared the non-differentiated growth of the hES cell line HS237, originally derived and thereafter cultured using human foreskin fibroblasts as feeder cells, by culturing it in media containing serum replacement (SR; 10, 15, 20%), FCS, and human serum. In addition, optimal concentrations of insulin-transferrin-selenium (ITS) mixture and the effect of basic fibroblast growth factor (bFGF) have also been studied. Cellular growth was monitored daily and maintenance of their non-differentiated character was studied using antibodies against TRA-1-60, TRA-1-81 and SSEA-4 and expression of Oct-4. The hES cells proliferated fastest when 20% of SR was used. In human serum-containing medium, the cells underwent extensive spontaneous differentiation within a few passages. The FCS supported the non-differentiated growth poorly. Basic fibroblast growth factor supported non-differentiated growth, the highest concentration (8 ng/ml) giving the best result, while ITS was not beneficial.