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Carbohydrate deficient transferrin (CDT) is produced by exposure to excess alcohol and is a marker of chronic alcohol misuse
Natalie Walsham MBiochem MSc DipRCPath
Department of Clinical Biochemistry, University Hospital Lewisham, London
Roy Sherwood BSc MSc DPhil
Department of Clinical Biochemistry,
King’s College Hospital NHS Foundation Trust, London, UK
Alcohol misuse is widespread among all socioeconomic groups worldwide with an estimated global lifetime prevalence of 16% in the adult population and is associated with significant morbidity and mortality. In the most recent report on alcohol from the World Health Organization (WHO, 2011) it was estimated that in excess of 70 million people worldwide had an alcohol use disorder (AUD) based on the classification in The Diagnostic and Statistical Manual of Mental Disorders (DSM-5) and that it causes 2.25 million or 3.8% of the total number of deaths in the world each year.1 The Health and Social Care Information Centre estimated that AUDs cost the National Health Service £3.5 billion per annum.2
Conventional methods for identifying subjects with an AUD have always relied upon the clinical history, examination or use of the AUDIT and CAGE questionnaires. However, deliberate under-reporting of alcohol consumption is a common problem with the use of these questionnaires. An ever-expanding range of biomarkers for the detection of harmful alcohol intake is available.3 These are typically divided into the direct (ethanol itself or specific metabolites), or indirect markers, the latter being changes in analytes due to the effect of alcohol or its metabolites directly or release of compounds consequent to the organ damage associated with excess alcohol intake.
Ethanol measurements in breath, blood or urine have a high specificity for excess alcohol intake, but because of the relatively rapid metabolism of ethanol, have a narrow time window for positivity. Other direct markers of alcohol intake are the consequence of alternative pathways of alcohol metabolism and include 5-hydroxytryptophol (5-HTOL) and more recently ethyl glucuronide (EtG) and/or ethyl sulphate (EtS).4 Indirect biomarkers include serum activities of enzymes released from the liver (for example GGT, AST), erythrocyte mean corpuscular volume (MCV) and carbohydrate-deficient transferrin (CDT).5 GGT is induced by alcohol itself and is also released from the liver due to hepatocyte damage caused by alcohol or its metabolites, thus reducing its specificity for alcohol misuse as it is increased in many non-alcohol-related liver diseases. The increasing prevalence of obesity and type II diabetes mellitus, resulting in hepatic steatosis, means GGT is often increased in individuals consuming little or no alcohol.6 MCV is raised in general ill-health and in specific nutritional deficiencies especially folate and vitamin B12 deficiency.
Definition and pathophysiology
Transferrin is a protein synthesised in the liver consisting of 679 amino acids of which the primary function is transportation of iron in the circulation, binding two ferric ions per molecule. It is glycosylated with two N-linked complex oligosaccharide side-chains each ending in a sialic acid residue (Figure 1). There is marked heterogeneity in these side-chains with each capable of being mono-, bi- and tri-antennary. Normal individuals have a preponderance of the tetrasialo-transferrin, which is predicted to have two bi-antennary side-chains attached to the 413 and 611 asparagine residues. Trisialo-transferrin and pentasialo-transferrin account for the remainder of the transferrin isoforms with the disialo-transferrin isoform being present in very small amounts (Table 1).
It was reported in the late 1970s that cerebrospinal fluid (CSF) and serum samples from alcoholic subjects had higher concentrations of the disialo-transferrin isoform, but it took a further 10 years for it to be recognised that this could be a potential test for excess alcohol intake.7,8 The pathophysiology of this is not fully understood, but is believed to be a combination of reduced biosynthesis and increased catabolism due to the direct effect of ethanol and/or its metabolite acetaldehyde on the enzymes involved in these metabolic pathways. In the ensuing years there have been various definitions for carbohydrate-deficient transferrin (CDT) which have included the asialo, monosialo, disialo and in some cases some or all of the trisialo-transferrin isoforms. It is now generally accepted that the monosialo-transferrin isoform has inherent instability, as modern analytical techniques do not detect it. Incorporation of the trisialo-transferrin isoform in CDT was predominantly to improve analytical sensitivity and resulted in a significantly worse clinical performance of CDT in most cases. In recent years there has been a move by the International Federation of Clinical Chemistry (IFCC) to reclassify CDT as simply the disialo-transferrin isoform.9
The sialic acid residues on the side-chains are necessary for recognition at the glycoprotein receptor responsible for the hepatic clearance of glycoprotein’s and conversely the disialo-transferrin isoform does not undergo accelerated clearance by the asialo-glycoprotein receptor resulting in a prolongation of its half-life in the circulation to approximately 10–14 days compared to normally glycosylated transferrin which has a half-life of about seven days.
Our understanding of the pathophysiology of the formation of CDT outlined above suggests two significant advantages of CDT over other biomarkers; (1) unlike GGT/AST CDT is formed by a direct effect of alcohol not organ damage so can be used in all but end-stage liver disease and (2) it is a marker of chronic heavy alcohol intake rather than a single session of excessive drinking.
The original methodology used by Stibler’s group7,8 was isoelectric focusing which is not a technique conducive to routine use. Subsequent methods have utilised separation of the isoforms by ion-exchange chromatography with analysis of the non-CDT and CDT fraction eluants by isotopic or non-isotopic immunoassays.10,11 Many of these methods suffered from analytical insensitivity and incorporated some or all of the trisialo-transferrin isoform in order to achieve reliable results; hence the percentage CDT in normal subjects was often quoted to be as high as 5%. For this reason, results from these early analytical methods need to be interpreted with caution.
Over the last ten years methods for the measurement of CDT have moved to techniques involving separation based on the size and charge of the molecule for example, high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). Figure 2 shows a schematic of the various isoforms of transferrin obtained using CE. Figure 3 shows the result of CE analysis for CDT in an individual whose alcohol consumption is within normal limits and whose CDT is <1.6%, whereas Figure 4 shows an electropherogram from a subject who is alcohol dependent with an increased CDT with both an increased disialo-transferrin fraction and the presence of asialo-transferrin. (NB: Figures 3 & 4 obtained using the SEBIA Capillarys CDT capillary electrophoresis method).
It is essential to report CDT as a percentage of total transferrin to avoid false negative or false positive results in subjects with either impaired hepatic synthesis or poor nutrition (low total transferrin leading to a false negative result) or up-regulated transferrin synthesis (high total transferrin leading to a false positive result) such as in pregnancy or iron deficiency.12
CDT formation requires sustained excess alcohol intake and could be regarded as the equivalent for alcohol misuse to the use of glycated haemoglobin in the assessment of glycaemic control in diabetics, with blood ethanol equating to blood glucose. CDT is not, therefore, significantly increased by a single episode of high alcohol intake or ‘binge’ drinking. EtG and/or EtS may prove the best markers for this group.4 CDT can be used in identifying individuals suspected of consuming excess alcohol but who deny it, in monitoring subjects who are participating in rehabilitation programmes or in workplace substance misuse testing schemes.
There have been a number of studies in healthy volunteers to attempt to ascertain the amount of alcohol required to increase CDT to pathological values and to determine the cut-off that achieves the best sensitivity and specificity for identification of AUDs. There is a general acceptance that an alcohol intake of more than 60g of ethanol a day for a 10–14 day period is required to increase CDT to more than 2%. From the many studies carried out in differing populations a range of sensitivities of 60–80% and specificities of 80–95% for CDT for the detection of alcohol misuse was obtained.13 Obviously, there is an inverse relationship between sensitivity and specificity depending on the cut-off chosen; lowering the cut-off increases sensitivity at the expense of specificity and vice versa. The choice of cut-off is therefore dependent on the particular requirements of the setting in which CDT is to be used. Additionally, if the use of only the disialo-transferrin isoform as CDT is adopted the cut-off applicable to the HPLC and CE methods may fall to 1.5–1.6%.
An example of where the cut-off selected is a conscious trade-off between sensitivity and specificity is in re-licencing of drivers classified as high-risk offenders (greater than twice the legal limit of alcohol for driving or a repeat offender). Since a study carried out for the Department of Transport in 2009 clearly demonstrated that GGT and MCV were affected by obesity, diabetes and chronic non-alcoholic liver disease, but CDT was not, the Driver Vehicle Licensing Authority (DVLA) in the UK have adopted CDT as the sole biological marker used in the decision to return a driving licence to high-risk offenders.6 The analytical method in use is the capillary electrophoresis method and a conscious decision was made to apply a cut-off of 2% rather than the clinical cut-off of 1.5%. Although this has the effect of reducing the sensitivity it increases the specificity providing reassurance that a positive result is more likely to be a true positive result. Attempts to improve the sensitivity and specificity of CDT have also included proposals for combinations of markers, typically CDT+GGT or CDT+MCV. In the main these combinations have resulted in slight improvements in sensitivity, but usually at the expense of the specificity.14
The high specificity of CDT for alcohol misuse is due to the relatively few causes of false positive results identified to date (Table 2). As CDT is hepatically cleared severe cholestatic liver disease for example, primary biliary cirrhosis, primary sclerosing cholangitis etc. results in retention of CDT, but this is clinically obvious as the serum bilirubin is usually markedly elevated. This does mean that CDT cannot be reliably used in monitoring patients on liver transplant waiting for abstinence from alcohol and EtG/EtS is the preferred marker in this clinical setting. Abnormal transferrin microheterogeneity has been reported in some patients with hepatocellular carcinoma, but our own studies have failed to replicate this. The congenital disorders of glycosylation (CDGs) are a theoretical cause of false positives. The CDGs are a family of disorders resulting from enzyme defects in the assembly or secretion of glycoproteins that usually present in childhood with severe neurological deficits and homozygotes present no diagnostic difficulty; data on heterozygotes is limited, but theoretically should have a CDT result between 25% and 50% which is well above values associated with alcohol misuse. Whilst drugs that can induce GGT synthesis number in the hundreds there are only a handful of case reports of drugs that could affect CDT.
CDT has been consistently shown to be a highly specific and reasonably sensitive marker of chronic alcohol misuse. It is not, however, a reliable marker of short-term or ‘binge’ drinking. Added to measurements of ethanol in body fluids and newer markers such as EtG/EtS it completes a family of markers covering a time window from four hours to 14 days that allows detection of AUDs.