Biomarkers of alcoholism: an updated review

Authors: S. K. Das ^a;  L. Dhanya ^a; D. M. Vasudevan ^a

Affiliation:

a Department of Biochemistry, Amrita Institute of Medical Sciences, Kerala, India

DOI: 10.1080/00365510701532662

Publication Frequency: 8 issues per year

Published in: Scandinavian Journal of Clinical and Laboratory Investigation, Volume 68, Issue 2 2008 , pages 81 - 92

First Published: 2008

Abstract

Introduction

Alcoholic beverages, and the problems they engender, have been familiar in human societies since the beginning of recorded history. Alcohol is no longer viewed as a threat to all people, but rather to a small subclass of “alcoholics” or, in today's technical terms, people who are “alcohol-dependent” 1. Measuring alcohol use and alcoholic liver disease in an individual or in a country has several limitations, and the definition of a “standard” drink varies significantly from country to country. Although most heavy drinkers imbibing more than 50 g per day do not develop significant liver disease, a certain amount is required to develop alcoholic liver disease (ALD) 2. Evidence suggests that the risk is increased with ingestion of >40 g/day for males (or about 4 drinks or two-thirds of a bottle of table wine) and >20 g/day for females 3. This level is considered “hazardous” and “harmful” in medical epidemiological meta-analyses 4. However, alcohol is causally related to more than 60 medical conditions 1.

Physicians have long sought an accurate and inexpensive means of identifying persons who consume excessive amounts of ethanol. It has been reported that medically diagnosed alcoholics can be differentiated from non-alcoholics using clinical laboratory tests. Moreover, distinguishing alcoholic from non-alcoholic liver disease patients has important implications for its treatment and management 5. Despite intensive investigation, there is still no satisfactory laboratory marker for surreptitious alcohol ingestion. Chronic alcoholism is diagnosed on the basis of clinical history, questionnaires about alcohol consumption and a number of laboratory investigations 6.

The perceived difficulty of obtaining an accurate drinking history may be one reason for the widespread under-diagnosis of alcohol misuse and related disorders 7. Ethanol can be easily and accurately measured in body fluids or vapours by several chemical and enzymatic methods. However, the existence of ethanol in the body is short lived and ethanol's metabolism and distribution within the body are extremely complex. The complex distribution and metabolism of ethanol and its metabolites can obscure the relationship between the ethanol concentration that is measured in body fluids or vapours and the amount that is actually taken into the body 8. Various blood tests have been used to aid in the assessment of drinking history. More recently, laboratory tests based on urine, breath and sweat analyses have been investigated, but there has been a great deal of controversy over the usefulness of these markers. Many conventional tests have only limited sensitivity and specificity, and there have been doubts whether there produce sufficient benefit to warrant their use 9.

Common laboratory tests

Patients with various forms of liver disorders show hyperbilirubinaemia 10. A significant increase in serum bilirubin levels, both unconjugated and conjugated, has been observed in alcoholic patients 11 12. Plasma urate level has been shown to correlate with recent alcohol intake. Heavy drinkers tend to have a slightly raised serum alkaline phosphatase (ALP; EC 3.1.3.1) 13. Urea concentrations are often reduced because alcohol inhibits enzymes in the urea cycle 14.

Gamma-glutamyltransferase

The most frequently used markers of alcohol intake are the serum enzymes gamma-glutamyltransferase (GGT; EC 2.3.2.2), aspartate aminotransferase (AST; EC 2.6.1.1) and the erythrocyte mean cell volume (MCV). Serum GGT is the most sensitive of these, and the most widely employed marker of alcohol consumption 15. The primary role of cellular GGT is to metabolize extracellular reduced glutathione (GSH), allowing for precursor amino acids to be assimilated and reutilized for intracellular GSH synthesis. This is a biliary canalicular enzyme which is induced by alcohol, and serum levels rise in response to acute hepatocellular damage. Levels are especially high in patients with severe alcoholic liver disease 16, though they may fall in the later stages of cirrhosis. When a heavy drinker is denied access to alcohol, any elevation of GGT should gradually resolve. Values fall to approximately half within 2 weeks, and usually return to the reference range over 6-8 weeks. This provides useful confirmation that alcohol was the cause of the elevation. However, the fall may be delayed or incomplete if there is underlying alcoholic hepatitis or cirrhosis or other medical disorders. It is more likely to be elevated in regular rather than episodic drinkers 15.

Significant differences were found in iso-GGT separated in sera from alcoholics and healthy subjects and patients with non-alcoholic liver disease. The iso-GGT fractionation is a complementary test in the diagnosis of alcoholic liver disease because of its high sensitivity 17. The iso-GGT pattern is probably due to the inductive action of alcohol or other agents 18. These raise some doubts as to the specificity of iso-GGT determination in the identification of alcoholic liver disease (ALD).

However, GGT is also raised in a variety of non-alcoholic liver diseases 19. High values are reported in subjects consuming barbiturates or other enzyme-inducing agents and in non-alcohol-related liver disease 20. It has moderate specificity. Medications and hepatobiliary disease may also cause elevation of GGT 21 22. A strong association of serum GGT with many cardiovascular risk factors and/or events might be explained by a mechanism related to oxidative stress 23. Other studies suggest that serum GGT might be an important predictor for developing metabolic syndrome and type II diabetes in middle-aged Japanese men 24.

Mean corpuscular volume

Alcohol and its metabolites have toxic effects on the production of haematologic precursor cells and on red cell morphology. Macrocytosis, enlarged erythrocytes, is a common finding in chronic alcoholics. Mean corpuscular volume (MCV) is an average estimate of the volume of erythrocytes and serves as an indicator of macrocytosis 25. If both GGT and MCV levels are elevated, it is likely that alcohol is the cause 26. Because the red blood cell survives for 120 days after it has been released into the circulation, MCV may remain elevated for up to 3 months after a person has stopped drinking. It is therefore less useful than GGT for monitoring alcohol intake in the weeks following treatment. An increase in MCV has been reported in conditions such as thyroid disease, folate deficiency, recent blood loss and a number of haematological conditions and other liver diseases. Anti-epileptics and non-alcoholic liver disease may also elevate MCV levels 22.

Transaminases

The serum transaminases - aspartate aminotransferase (AST) and alanine aminotransferase (ALT) - are less often elevated than GGT 27. They are therefore seldom used in screening programmes because of their limited sensitivity 22 28. Serum AST can also arise from non-hepatic sites, particularly heart and muscle, and levels are increased in conditions such as myocardial infarction and skeletal muscle trauma. The ratio of AST to ALT in serum may help in the diagnosis of some liver diseases. In most patients with acute liver injury, this ratio is 1 or less, whereas in alcoholic hepatitis it is generally about 2 29. Deficiency of pyridoxal-5'-phosphate, a necessary coenzyme for both aminotransferases, is common in alcoholic liver disease. This deficiency decreases hepatic ALT to a greater extent than AST, with corresponding changes in serum concentrations 30.

In the serum of healthy individuals, mitochondrial AST (mAST) makes up only <10 % of total AST activity 31. However, following heavy alcohol consumption there is evidence of mitochondrial damage, with an increase in the proportion of mitochondrial AST to total AST (mAST: tAST) in serum 32. A high ratio in the serum of mitochondrial to total AST indicates mitochondrial damage and provides further evidence of alcoholic liver disease 30.

AST to platelet ratio index (APRI)

AST level elevates and platelet count reduces in ALD patients. This observation has been extrapolated to a new index known as AST to platelet ratio index (APRI). APRI may be useful in predicting the fibrosis stage 33. However, it is not an independent predictor of significant fibrosis in the best-fitting multivariate model 34. The correlation coefficient of APRI with stage of fibrosis is poor. In addition, GAPRI (GGT to platelet count) and HAPRI (hyaluronic acid to platelet count) and several other ratio indexes are being tested as fibrotic indices 35.

Albumin and globulin

A common feature of chronic alcoholic liver disease is progressive hypoalbuminaemia 29 36 37. Acute exposure to alcohol depresses albumin. Despite a rise in mRNA level of albumin in liver in response to alcohol intoxication 36, the decrease in serum albumin level is attributed to the nutritional status of the subjects 38 39. However, the albumin is a potential subject of formation of adduct by acetaldehyde, an alcohol metabolite. This albumin or other protein adducts can stimulate the formation of immunoglobulins, thus causing a rise in serum globulin level 40. Nomura et al. 41 identified two proteins (5.9 and 7.8 kDa) in alcoholic patients. The 5.9 kDa protein was identified as a fragment of the fibrinogen alphaE chain and the 7.8 kDa a fragment of apoprotein A-II. These novel protein fragments may be promising biomarkers for excessive alcohol drinking 41. Ethanol consumption slows down the rate of hepatic protein catabolism. Such changes may be related to the degree of ethanol-induced oxidative stress 42.

Acetaldehyde

Acetaldehyde has also been used as a marker of recent drinking. Since acetaldehyde is a reactive molecule, forming Schiff bases with amines, it readily binds to proteins, leading to an irreversible reaction giving an acetaldehyde-protein adduct 43. Two approaches have been taken in the detection of acetaldehyde as a marker of alcohol intake. The first is to detect acetaldehyde, which is free or reversibly bound to plasma proteins or to blood cells 44. The acetaldehyde is liberated from the blood and measured by gas or liquid chromatography 45. The second is to use an immunoassay to detect epitopes derived from acetaldehyde on proteins in the plasma 46 47. Neither approach has been examined sufficiently for its value in detecting and monitoring alcohol consumption to be fully determined, but each shows promise. It is not yet clear whether the adducts between acetaldehyde and liver proteins are released as a result of liver damage or are themselves involved in inducing immunological damage 46. Antibodies to acetaldehyde-modified epitopes have also been used as markers of recent alcohol intake 48. In particular, the IgA response to acetaldehyde-modified epitopes has been reported to be a specific marker of alcoholic liver disease 49.

Haemoglobin associated acetaldehyde (HAA)

Haemoglobin is another protein that forms adducts with acetaldehyde after ethanol ingestion 28 50. HAA formation peaks 30 min post-alcohol ingestion, because the reactive acetaldehyde, generated by the action of oxygenated haemoglobin on alcohol, initially forms a reversible adduct with erythrocyte haemoglobin 51-53. These HAA reversible complexes, detectable in the blood stream for up to 48 h after the last drink, are converted to irreversible HAA 53. The irreversible HAA accumulates with time and remains detectable in the bloodstream for at least 28 days 51 52.

HAA levels in males are greater than those found in females, but the gender difference is explained by the sex-dependent haemoglobin concentrations 52 54 55. HAA levels in alcohol abusers have been found to be significantly higher than levels found in teetotalers 52 54. HAA increases significantly with alcohol ingestion, even after a single high dose of ethanol (2 g/kg), when the conventional markers, GGT and MCV, show no change. HAA has high specificity and serves as a good marker of alcohol abuse in women 28. HAA has been found to have better sensitivity and specificity than GGT, ALT, AST and MCV in one alcohol treatment programme 55.

Dehydrogenases

Aldehyde dehydrogenase (ALDH) is the principal enzyme involved in the oxidation of acetaldehyde. Alcoholic and non-alcoholic patients with similar degree of liver pathology show no difference in total ALDH activity. However, total ALDH activity in cirrhotics is significantly lower in alcoholic and non-drinking cirrhotics than in controls. The isoenzyme found in the erythrocyte resembles the hepatic cytosolic isoenzyme and has a low affinity for acetaldehyde 56. While activity levels are reduced in recently drinking alcoholics, there is a degree of overlap with control values 57, which limits its clinical usefulness.

Increased acetaldehyde production via alcohol dehydrogenase (ADH) has been implicated in the pathogenesis. ADH is an autoantigen in autoimmune liver disease and in a proportion of patients with alcoholic liver disease. Ma et al. 58 suggested that anti-ADH antibodies might be triggered by alcohol consumption and act as a disease activity marker in alcoholic liver disease.

Lipid profile

High-density lipoprotein cholesterol (HDL-C) levels correlate with recent intake; however, the sensitivity of an abnormal HDLC in detecting alcohol misusers is limited 59. Alcohol misuse might result in increased inflammation leading to oxidation of low-density lipoprotein cholesterol (LDL-C) 60. Non-oxidative metabolism of ethanol to fatty acid ethyl esters (FAEEs) is an important pathway of ethanol disposition during chronic alcohol abuse, and FAEE concentrations can be a more reliable biomarker of chronic alcohol abuse than a history of acute alcohol abuse 61. Pragst et al. 62 suggested that FAEE in skin surface lipids could be used for medium-term retrospective detection of heavy drinking. FAEEs are formed in almost all human tissues, including blood after alcohol consumption, and incorporated from sebum into hair, where they can be used as long-term markers for excessive alcohol consumption. Wurst et al. 63 suggested that FAEE is a potentially valuable marker of chronic intake of high quantities of ethanol. Furthermore, a reasonable and provisional FAEE cut-off to distinguish between social/moderate and heavy drinking/alcoholism in hair is 0.4 ng/mg 63.

Total serum sialic acid

The measurement of total serum sialic acid (TSA) has been proposed to serve as a marker for excessive alcohol consumption 64. Most of the sialic acid found in blood is conjugated and resides at terminal positions on carbohydrate side chains of proteins. TSA has been measured by two approaches. The first is to hydrolyze conjugated sialic acid residues from serum proteins with strong acid or the enzyme TSA neuraminidase. The second is to hydrolyze protein conjugated sialic acid residues in serum samples, purify sialic acid, and quantify using high performance liquid chromatography (HPLC) 64. While these methods are not complicated and could be performed in most research laboratories, they are not commonly available throughout clinical laboratories. Certain forms of cancer 65, cardiovascular disease 66 and diabetes 67 result in the elevation of sialic acid residues in serum. TSA levels in serum have also been shown to vary with body mass index, age and blood pressure among both men and women 68.

Plasma sialic acid index of apolipoprotein J

Apolipoprotein J (Apo J) is a glycoprotein of MW 70 kDa that is found in high-density lipoprotein complexes (HDL2 & HDL3). It has two non-identical, disulphide-linked subunits. Apo J has an isoelectric point of approximately 5.0 and contains about 30 % carbohydrate. The physiological significance of Apo J is not well understood, but it is thought to be involved in the exchange of lipids, especially cholesterol, between different lipoproteins 69. An advantage in studying the level of sialylation of Apo J as a marker for alcohol consumption is that human and rat Apo J have been shown to have 28 mol of sialic acid per mole of Apo J 70, a large number compared to six sialic acid residues on transferrin. The term sialic acid index of Apo J (SIJ) has been coined to express the ratio of moles of sialic acid per mole of Apo J protein. Preliminary studies indicate that SIJ levels are reduced with heavy, prolonged alcohol intake and that they return to normal levels over a period of weeks, with an approximate half-life of 4 to 5 weeks 70 71. Levels of liver sialyltransferases were reduced and levels of liver sialidase were elevated in a proportional manner to SIJ. Sialyltransferase catalyzes the addition, while sialidase catalyses the removal of Apo J sialic acid residues 70.

Carbohydrate-deficient transferrin

Transferrin (Tf) (siderophilin) is a globular protein which is responsible for iron transport in plasma 72 73. It has MW 79.5 kDa and consists of a single polypeptide chain of 679 amino acids arranged in two independent metal ion-binding globular domains, the N-terminal (amino acids 1-336) and the C-terminal (amino acids 337679), and two N-linked complex glycan chains located in positions 413 and 611 74. The two Tf N-glycan chains differ in their degree of branching, showing bi-, tri- and tetra antennary structures. Each antenna of the Tf N-glycan chains terminates with a negatively charged sialic acid molecule. Because of this, asialo-Tf and the sialylated forms monosialo through octasialo-Tf can occur in serum 75. The carbohydrate represents about 6 % of the transferrin molecule 76.

Tf reversibly binds numerous polycations - iron, copper, zinc, cobalt and calcium - although only iron and copper binding appears to have physiological significance. Each transferrin molecule has the capacity to combine with two atoms of ferric ions and an associated anion, which is, in vivo, usually bicarbonate ion 77, this binding is a pH-dependent process 78. The two carbohydrate (Tf N-glycan) structures bind a maximum of six sialic acid residues.

Consumption of heavy alcohol is associated with a significant elevation in the risk of iron overload 79; 20-30 % of alcohol abusers have increased liver iron stores 80. Iron accumulation occurs among alcoholics as a result of increased intestinal mucosal permeability 81. It is well known that alcohol promotes the absorption of ferric iron from the gastrointestinal tract, possibly by inducing gastric secretion of HCl 82. Tf plays an important role in iron homeostasis by attaching to the receptors on the cell surface before iron is released into the cell. After alcohol intake, an increase in iron level and, secondarily, a decrease in transferrin synthesis occur.

Carbohydrate-deficient transferrin (CDT) is one of the conventional biochemical markers of chronic alcohol misusers used by researchers and clinicians. CDT is a collective term referring to isoforms of transferrin which are deficient in sialic acid residues, corresponding to asialo-Fe Tf, monosialo-Fe Tf and disialo-Fe Tf 83. A number of enzymes are affected by ethanol intake. The induction or inhibition of sialyl transferase and plasma sialidase may be involved in the CDT level elevation. An alteration of protein transport during post-translational modification could be a primary mechanism in the impairment of protein metabolism associated with chronic alcohol abuse 84. CDT has been approved by the FDA as a clinical diagnostic test for the detection of heavy alcohol consumption 85.

Most patients with liver disease have normal CDT 83 86. Advanced chronic liver disease (primary biliary cirrhosis, chronic active hepatitis and drug-induced hepatic insufficiency) causes false-positive results 83. CDT tends to have high sensitivity and specificity in distinguishing chronic, heavy drinking subjects from abstainers or very light social drinkers 86-88. Accuracy for this marker is generally poorer among drinkers with a low level of alcohol consumption 89, younger alcoholics 90 91 and women 92 93. Interestingly, the sexes seem to differ in which isoform types are increased by alcohol. There is no consensus about the pattern of drinking needed to elevate serum CDT values, not at least in patients who are alcohol-dependent 88.

Transferrin being a steroid responsive protein, sex-based hormonal variations might contribute to the lower sensitivity of CDT. Varying hormonal status such as pregnancy, use of contraceptives, menopause/menstrual cycle can alter iron homeostasis in women. CDT levels are markedly affected by iron homeostasis. Several CDT assay methods appear promising, but it is not readily apparent which is the most accurate 84. Quantitative evaluation of transferrin fractions is complicated 94. Whatever the method used for CDT detection, a huge prerequisite is its precision and accuracy.

Although CDT is usually unaffected by the presence of liver disease, several conditions have been found to affect serum CDT concentrations 84. One reason for high CDT concentrations, despite normal alcohol consumption, is the carbohydrate-deficient glycoprotein (CDG) syndrome, a group of autosomal recessive diseases, the defining feature of which is a carbohydrate defect in serum Tf. Tf-B (“busy”) and Tf-D variants can interfere with CDT analysis. However, false-positive results can also occur because of genetic D-variants of transferrin, primary biliary cirrhosis, hepatocellular carcinoma, viral liver cirrhosis, pancreas and kidney transplantation or the drugs used to treat these disorders 95. Therefore clinical interpretation of CDT needs careful assessment in patients with alcohol-related or non-alcohol-related health disorders. The laboratory should report the CDT value, the cut-off value and the method of analysis. Changing the CDT test can cause a sharp increase or decrease in CDT values, which can lead to actual drinking status being misinterpreted.

Serum beta hexosaminidase

Beta hexosaminidase (beta-HEX; also called N-acetyl beta glucosaminidase) is a lysosomal hydrolase that exists in most cell types and is involved in the metabolism of carbohydrates and gangliosides in liver cells. The beta HEX molecule is composed of combinations of two polypeptide chains, termed alpha and beta, which results in the existence of several isoforms of beta-HEX (B, I, P, A and S). Isoforms B, P and I contain two beta subunits, isoform A is composed of alpha and beta subunits, while isoform S contains only alpha subunit 96. Isoforms B, I and P are heat stable, while isoforms A and S are heat labile 97.

The heat stable isoforms of beta-HEX (B, I, P), collectively called beta-HEX B, have been elevated in serum and urine 98. After heavy alcohol consumption, lysosomes are damaged and subsequently cells release the enzyme into the blood 99 100. Serum beta-HEX activity as a percentage of the total beta-HEX activity, which is called beta-HEX B %, is the most sensitive approach 100.

Elevated serum beta-HEX occurs in patients with cholestasis and cirrhosis, due mainly to increases in beta-HEX P isozyme 96 101, in hypertension, diabetes mellitus, myocardial infarction, thyrotoxicosis, pregnancy 98 and slightly with age 98 102. Reduced serum beta HEX levels have been seen in association with chronic renal failure 98.

Hyaluronic acid

Serum hyaluronate (hyaluronic acid; HA) concentrations increase in various liver diseases, especially in alcoholic liver disease, and serum HA concentration has been used as a marker for hepatic fibrosis 103. However, it is unknown whether or not hepatic HA contents in ALD are increased by alcohol. Hepatic HA contents in ALD may be increased by alcohol in addition to hepatic fibrosis, and increased HA deposition in the liver may be reversible by abstinence of alcohol.

Ethyl glucuronide

Ethyl glucuronide (EtG) is a non-volatile, water-soluble, stable, direct metabolite of ethanol that can be detected in various body fluids, tissues and hair. Shortly after the consumption of ethanol, even in small amounts, EtG becomes positive. It can detect ethanol intake up to 80 h after complete elimination of alcohol from the body, i.e. covering a unique and important time spectrum for recent alcohol use. EtG seems to meet the need for a sensitive and specific marker to elucidate alcohol use that is not detected by standard testing. EtG is a useful tool in numerous settings, including alcohol and drug treatment 104. EtG offers an extended window for assessment of drinking status (up to 5 days) 105. After moderate drinking, EtG in the urine has proved to be a superior marker of recent ethanol consumption in healthy subjects 106.

Blood alcohol concentration

The bloodstream transports ethanol to all parts of the body 107, such that most tissues are exposed to the same concentrations as in the blood 108. The vascularity of tissues influences the distribution of alcohol, and their water content determines the amount of alcohol present after equilibrium 109. The rate of equilibration is governed by the ratio of blood flow to tissue mass. Arterial and venous concentrations differ as a function of time after drinking. Ethanol has low solubility in lipids and does not bind to plasma proteins, so volume of distribution is closely related to the amount of water in the body, contributing to sex-related and age-related differences in disposition 107. Ethanol is eliminated predominantly (95-98%) by hepatic metabolism and the remainder is excreted in breath, urine or sweat 107 108. A very small proportion (less than 0.5 %) is also excreted in tears 110. Therefore, blood alcohol level is a reliable measure only when blood is sampled within 24 h of alcohol consumption 22. A positive blood alcohol concentration (BAC) provides a highly specific indication of recent drinking. Breath-analysers provide an immediate result, the levels correlating well with blood alcohol. Urine alcohol gives an accurate indication of the BAC at the time the urine was produced. Transdermal alcohol sensors or sweat patches are promising as a means of prospectively measuring alcohol intake over several days 111 112. Because the BAC detects alcohol intake in the previous few hours, it is not necessarily a good indicator of chronic excessive drinking. BAC-based pre-evaluation before testing chronic alcohol abuse among drivers is most relevant in a traffic safety context 113. Most countries have a permissible limit of 0.2 to 0.5 promille for traffic security 114.

Urine examination

The serotonin metabolite 5-hydroxytryptophol (5HTOL) is a normal, minor constituent of urine and is excreted mainly in conjugated form with glucuronic acid. The formation of 5HTOL increases dramatically after alcohol intake, due to a metabolic interaction, and the elevated urinary excretion remains for some time (>5-15 h depending on dose) after ethanol has been eliminated. This biochemical effect can be used in the detection of recent alcohol intake. An elevated urinary 5HTOL level can serve as a sensitive and reliable marker for recent alcohol intake with a number of clinical and forensic applications 115. The urinary ratio of the serotonin metabolites, 5-hydroxytryptophol (5HTOL) and 5-hydroxyindoleacetic acid (5HIAA) has been found to reflect alcohol intake in the past 24 h 116. As the test measures very recent alcohol intake, its clinical use in monitoring a patient's intake requires frequent urine collection.

The assays of urinary alanine aminopeptidase, the enzyme released from the brush border membranes of renal proximal tubules, relate to the nephrotoxic effects of alcohol abuse and could be a valuable complement to the other presently used markers of chronic alcohol abuse that are generally based on ethanol hepatotoxicity 117.

Type IV collagen

Chronic liver disease is a series of progressive disorders culminating in liver cirrhosis and characterized by excessive amounts of deposited collagen. Serum type IV collagen derives from the type IV protocollagen pool and is a sensitive marker for the fibrogenetic process in hepatic basement membranes 118. Although various types of collagen (types I, III, IV, V and VI) increase proportionally in the liver with the progression of fibrosis, type IV collagen, a constituent of the basement membrane, is particularly noteworthy for the following reasons: it relates to aggravation of hepatocellular damage and hepatocellular dysfunction, it is important in hepatocellular regeneration and rearrangement of the loblular architecture, and it is the earliest type of collagen to be synthesized by hepatocytes in experimental liver injuries. Determination of serum type IV collagen and laminin are useful in the diagnosis of hepatic fibrosis, and a combination of the two parameters is recommended 119. Serum type IV collagen levels in the group with alcoholic cirrhosis showed significantly higher values than the others in one study 120. We also reported that serum type IV collagen level increased significantly in alcoholic liver disease patients compared to non-alcoholic fatty liver disease patients 121. Compared with other markers, serum concentration of type IV collagen may more strongly reflect the histologic features of ALD 122.

Cytokines

Alcoholic liver disease (ALD) patients have a significantly higher number of Kupffer cells, which enhances expression of extracellular matrix and promotes fibrogenic processes 123. Activation of Kupffer cells by gut-derived endotoxin plays a pivotal role in alcoholic liver injury, although it has been reported that acute ethanol administration reduces activation of Kupffer cells. Kupffer cells isolated from rat treated only once with ethanol are sensitized to endotoxin 24 h later correlatively with CD14 expression 124. Moreover, Kupffer cell activation by endotoxin via Toll-like receptor (TLR)-4 is involved in alcohol-induced liver injury and ethanol-induced oxidative stress is important in the regulation of transcription factor NFkappaB activation and cytokine production by Kupffer cells 124.

Increased exposure of Kupffer cells to lipopolysaccharide (LPS) during chronic ethanol exposure leads to the production of a number of inflammatory mediators, including tumour necrosis factor alpha (TNF-alpha). Early growth response-1 (Egr-1), an immediate-early gene transcription factor, is an important contributor to increased LPS-stimulated TNF-alpha secretion by Kupffer cells after chronic ethanol exposure. In other models of tissue injury, such as ischaemia reperfusion in the lung, Egr-1 acts as a coordinator of the complex response to stress. In addition to the critical role of Egr-1 in generating maximal LPS-stimulated TNF-alpha expression, Egr-1 also controls the expression of a number of inflammatory mediators, including intercellular adhesion molecule (ICAM)-1, monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-2, as well as genes contributing to fibrosis, such as transforming growth factor (TGF)-beta1, platelet-derived growth factor (PDGF)-A chain and fibroblast growth factor (FGF) 125.

TNF-alpha constitutes a major factor in the development of alcohol-induced liver injury. In alcohol-dependent subjects, elevated levels of plasma TNF-alpha are strongly predictive of mortality. Binding of TNF-alpha to TNF-alpha receptor-1 (TNF-R1) activates death domain pathways, leading to necrosis and apoptosis in most tissues, and increases the expression of intercellular adhesion molecules (i.e. ICAM-1), which promote inflammation. Exposure of different cell types to pharmacologic concentrations of ethanol increases TNF-R1 levels and may augment TNF-alpha-mediated cell injury in different tissues 126. TNF-alpha makes a moderate contribution to the ALT elevation, necroinflammation, apoptosis, a small contribution to the fatty liver and no contribution to hyperhomocysteinemia and endoplasmic reticulum stress in intragastric alcohol-fed mice 127.

Excessive alcohol consumption is associated with the generation of antibodies against neoantigens induced by ethanol metabolism. Alcoholic liver disease is associated with the generation of IgAs and IgGs against acetaldehyde-derived antigens and enhanced levels of both pro- and anti-inflammatory cytokines, whereas elevated IgA, IL-6 and IL-10 characterize alcoholics without liver disease 128. Interleukin (IL)-8 is activated in alcoholic liver disease, especially in alcoholic hepatitis, and is closely correlated with liver injury. IL-8 levels can reflect the stage and severity of alcoholic liver disease, and may serve as a predictor of survival in patients with alcoholic hepatitis 129. Exceptionally high concentrations of serum carbohydrate antigenic determinant, CA 19.9, may be found in patients with alcoholic liver disease 130, while ethanol intake may act, in part, on the increase of serum des-gamma-carboxy prothrombin (DCP) in ALD 131. 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.

Oxidant-antioxidant system

Alcohol consumption is associated with a number of changes in cell functions and the oxidant-antioxidant system 132. Reactive oxygen species are significantly higher in heavy drinkers than in controls. The total antioxidant capacity is similar in chronic alcohol abusers and in moderate drinkers. Oxidative stress can be observed in heavy drinkers without severe liver disease 133. Enhanced microsomal malondialdehyde formation, a lipid peroxidation index and a decreased level of the antioxidant, alpha- tocopherol, accompanies the stimulation of hepatic cytochrome P-450 monooxygenase activity. Thus, the level of malondialdehyde and alpha-tocopherol in the serum may be recommended as biological markers of ethanol-provoked oxidative stress 134. Plasma 8-isoprostane may increase in habitual drinkers due to the oxidization stress induced by alcohol intake, and become a useful marker for estimating the drinking habit 135. Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) has been reported as a sensitive biomarker of oxidative DNA damage and also of oxidative stress. Drinking and smoking-induced liver damage by oxidative stress and urinary 8-OHdG may be reliable markers of oxidative stress in patients with chronic liver disease 136.

Peroxidative degradation of lipids yields the aldehyde 4-hydroxy-2-nonenal (4HNE) as a major product. Uncontrolled oxidative stress can yield excessive lipid peroxidation and 4HNE generation, however, and overwhelm these cellular defences. Recent evidence suggests a role for protein modification by 4HNE in the pathogenesis of several diseases (e.g. alcohol-induced liver disease); however, the precise mechanism(s) is currently unknown, but likely results from the adduction of proteins involved in cellular homeostasis or biological signalling 137. Many enzymes or proteins involved in stress response, chaperone activity, intermediary metabolism and antioxidant defence systems such as peroxiredoxin are oxidized after alcohol treatment. Many oxidized proteins after alcohol exposure are degraded. Kim et al. 138 developed a sensitive method using biotin-N-maleimide (biotin-NM) as a probe for positively identifying oxidized mitochondrial proteins.

Conclusions

Several blood tests have been used as composite indices in the detection of hazardous alcohol consumption, sometimes aided by computer algorithms 5 139 140. While this provides higher sensitivity than any one test alone, it involves increased expense and complexity, and has not become routine practice. Most laboratory tests have been evaluated for their ability to detect subjects dependent on alcohol. The level of intake at which laboratory results become abnormal will vary from person to person. Some subjects with hazardous intake will have normal test results, and some with “non-hazardous” intake will have elevated results. Several laboratory tests can also be used to assess the presence and extent of physical harm resulting from excessive consumption.

Research over past years has revealed several markers with considerable potential for more accurate reflection of recent alcohol intake. These include CDT, total serum sialic acid (TSA), sialic acid index of Apo J (SIJ) beta-hexosaminidase, acetaldehyde adducts and 5HTOL: 5HIAA. These markers provide hope for more sensitive and specific aids to diagnosis and improved monitoring for intake. Several biochemical and haematological tests, such as gamma glutamyltransferase (GGT), aspartate aminotransferase (AST) and erythrocyte mean corpuscular volume (MCV) are established markers of alcohol intake 141, but lack sensitivity when used singly 142. Their validity as markers is based largely on correlations with recent intake at a single time-point and on decreases in elevated values when heavy drinkers abstain from alcohol. These readily available laboratory tests provide important prognostic information and should be integral parts of the assessment of persons with hazardous alcohol consumption.

“To find a man's true alcohol intake, you double what he says and halve what his wife says.” - Anonymous.

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