The carbohydrate-deficient transferrin as a marker of chronic alcohol consumption and assessment of its usefulness in patients with cardiovascular diseases – a pilot study

Cardiovascular diseases (CVD), including ischemic heart disease, ischemic stroke, and peripheral arterial disease, are, next to cancer, the most common cause of death both in Poland and worldwide. WHO estimates that 16.6 million people die every year due to cardiovascular disease, and 600 million are in the so-called group of increased risk of death. In Europe, the number of deaths due to CVD is estimated at 4.3 million. Both genetic predispositions and environmental factors have an important influence on the development of cardiovascular diseases [1,2]. A risk factor for cardiovascular diseases is considered to be any measurable trait the occurrence of which in the population of healthy people is associated with a statistically significant increase in morbidity or death frequency from CVD [3,4,5]. The INTERHEART study, conducted in 32 countries, clearly indicated the importance of “classical” risk factors as responsible for the development of CVD, such as dyslipidemia, smoking, hypertension, diabetes, and abdominal obesity [1,6,7]. Moreover, adverse psychosocial factors, inadequate diet, excessive alcohol consumption, lack of regular physical activity, aging, and gender may also influence general cardiovascular status. CVD biomarkers can detect pathological changes at a very early stages of the disease, and thus are suitable for use in the determination of short- and long-term risks of cardiovascular events. The intensive development of methods used in clinical biochemistry has allowed the identification of many new, promising markers which are so-called new risk factors and markers: inflammatory biomarkers (hs-CRP, fibrinogen), thrombotic biomarkers (homocysteine, Lp-associated phospholipase A2), and specific organ markers (N-terminal fragment brain natriuretic peptide, troponin T) [8,9,10,11,12].

The guidelines of the European Society of Cardiology (ESC) on the prevention of CVD, published in 2019, recommend especially healthy lifestyle modifications and reducing alcohol consumption to a minimum. The Sixth Joint Working Group of the European Society of Cardiology and other scientific societies on CVD prevention in clinical practice have confirmed that excessive alcohol consumption is associated with increased mortality influencing progress of CVD. Ischemic disease mortality was found to be higher among men (65%) who consume significant amounts of alcohol. Among these diseases, heart failure occupies a special place. Heart failure causes over 100,000 deaths annually in Europe. Heart failure is a condition in which the heart is unable to pump enough blood to cover the tissue needs due to impaired structure or function, or by increasing the filling pressure [13]. A decrease in the strength of the heart's contraction can be caused by a number of diseases that lead to heart muscle damage. One of the most common causes is ischemic heart disease, which reduces the oxygen supply to the heart muscle due to the presence of atherosclerotic plaques and thrombotic events in coronary vessels, leading to myocardial infarctions and loss of contractile functions of the left ventricle. Other causes of heart failure include hypertension, arrhythmias, myocarditis, heart valve defects, and cardiomyopathies (congenital heart defects). Cardiomyopathies are one of the more common causes of sudden cardiac death or heart failure, and in some advanced heart failure patients they are an indication for heart transplantation. Heart failure often affects young people of productive age, which significantly affects and increases the cost of treatment. Guzzo-Merello et al. [14] emphasized the importance of chronic alcohol consumption in the development of cardiomyopathy.

Numerous studies showed the adverse effects of alcohol (ethanol) consumption on the heart and circulatory system. With alcohol abuse—over (60 g alcohol/day) —an increased risk of arterial hypertension and the development of cardiac arrhythmias, hemorrhagic stroke, and cardiomyopathy are observed [11,15,16].

Paradoxically, so far scientific societies dealing with the problem of alcohol consumption have not developed uniform recommendations regarding the acceptable levels of alcohol consumption versus an increased risk of development of cardiovascular diseases [17,18,19].

Desialized transferrin as a marker of chronic alcoholism showed a high prognostic value and enables the identification of groups of patients who do not comply with medical recommendations, thus contributing to the optimization of treatment costs.

Alcohol markers can be a very useful screening tool among general practitioner's patients, in emergency rooms and internal medicine departments, as well as in the assessment of the impact of alcohol in the disease progression, and diagnosis or confirmation of the effectiveness of the therapy [20]. Detection of increased levels of desialized transferrin could also be valuable within the heart transplant qualification process since alcohol use disorder may also be one of the contraindications for this treatment. The goal of the study was to confirm the diagnostic value of desialized transferrin among patients with heart failure as measured by N-terminal fragment brain natriuretic peptide (NT pro-BNP), troponin T, gamma-glutamyltransferase (GGT), and alkaline phosphatase (ALP) levels.

Asialo-, disialo-, trisialo-, tetrasialo-, pentasialo-transferrin were eluted at retention times of 4.08, 7.10, 8.00, 10.12, and 11.20, respectively, and peaks obtained fulfilled validity criteria according to shape and separation. The specificity of the method was confirmed using serum samples randomly taken from 20 healthy patients from the control group. No interfering peaks were observed in chromatograms from patient samples and quality controls. The representative chromatograms are shown in Figure 1.

Figure 1.

Measurements of CDT, NT pro-BNP, troponin T, ALP, and GGT were available for 59 patients.

Descriptive statistics for CDT and other analyzed parameters are presented in Table 1. Mean CDT in overall group was 1.5 (±0.9). Six (10.2%) patients had CDT over 2.5 and 40 (6.7%) over 1.2.

Analysis of correlation between CDT and other parameters revealed significant positive, yet not very strong association between CDT and ALP and GGT (both coefficients 0.327, p<0.05; Figure 2).

Figure 2.

In the next step, ROC curve analysis was performed to assess ability of NT pro-BNP, troponin T, ALP, and GGT parameters to detect patients, for which CDT is over 2.5 and over 1.2 (Figures 3 and 4).

Figure 3.

Figure 4.

The area under the ROC curves along with its 95% confidence interval for each curve is presented in Table 2.

For the cut-off point 2.5, the only variable that can be considered to have predictive power was GGT; for cut-off point 1.2, the variable that can be considered to have predictive power was NT pro-BNP (as lower limit of 95% CI is over value 0.500). For GGT and CDT, the cut-off of 2.5 value of 286 was selected as the best threshold resulting in sensitivity 88.8% and specificity 83.3%. For cut-off 1.2, the best threshold for NT pro-BNP was 2662.5 resulting in sensitivity 84.2% and specificity 60.0%.

Desialized transferrin was found to increase significantly after a minimum weekly consumption of at least 50–80 g of alcohol per day. During the period of abstinence, values become normalized within 14–17 days, which is the mean half-life of CDT. Interestingly, consumption of even small amounts of ethanol during the period of abstinence leads to a re-increase of CDT levels in sera. The concentration of desialylated transferrin returns to physiological values after a few weeks of discontinuing the consumption of ethyl alcohol [23]. The application of specific analytical tool capable to monitor of alcohol consumption is highly expected as alcohol consumption is constantly increasing and covers as much as 80% of the population in Poland. We can, however, suspect that patients’ consumption of alcohol might be lower than observed in the general population, as patients with different disorders are generally more caring about maintaining a healthy lifestyle.

It is assumed that the lack of a clear position or recommendation from medical authorities on the allowed alcohol uptake issue may be related to the not fully explained but frequently followed belief in the so-called “beneficial” effects of small amounts of alcohol, which unfortunately can lead to addiction. Nevertheless, chronic alcohol consumption is a problem commonly recognized worldwide, not only in Poland, resulting in many cases in irreversible health damages, especially in patients under medical therapy. During continuous alcohol consumption, a substance belonging to the iron-transport group of glycoproteins named transferrins with a reduced amount of sialic acid, called a marker of chronic alcoholism, appears in the body within 7 days, thus can be potentially detected in each suspected patient. Chronic alcohol abuse causes disturbance of the transferrin biosynthesis or tissue damage resulting the presence of asialo-transferrins among more than about 3% disialo-transferrins. Due this processes desialylated transferrin has a reduced amount of sialic acid (asialo-, monosialo-, and disialo-transferrin) [24]. So, excessive consumption of alcohol contributes to an increase in the amount of low-carbohydrate isoforms of so-called desialylated transferrins, which under the physiological conditions of the body occur at a very low level. Thus, ethyl alcohol and its metabolites affect the functioning of transferrin-modifying enzymes, limit the attachment of sialic acid to transferrin molecules and lower formation of desialized forms, and disrupt the functions of hepatocyte receptors responsible for the elimination of desialylated transferrin [25].

We found a high incidence of the CDT above 2.5%, especially in a patient with high GGT. Higher than 1.3% percentage of desialylated transferring detected in the study group is considered as the result of the so-called “gray area”, which means that these heart failure patients should be carefully monitored for possible alcohol consumption despite negative screening psychological tests. However, the scale of the health risk is greater in patients when the results coexist with other factors (e.g., GGT, ALP biomarkers).

ALP and traditional marker of chronic alcohol abuse γ – GGT are located in the bile and are related with some type of damage of biliary canaliculi. ALP has been reported as a rather novel risk marker (inflammatory mediator) for cardiovascular disease, and GGT is regarded as a sensitive indicator of hepatocyte damage. GGT or ALT increased serum activity is mainly observed as a result of the intensified biotransformation of xenobiotics, mainly ethyl alcohol [23,25,26].

Salaspuro [26], in a study comparing the value of CDT to other laboratory markers (e.g., GGT), found a high relevance of CDT. Their ROC analysis confirmed that CDT was the best biological marker, although its CDT-ROC curve did not differ statistically significantly from that of the conventional laboratory markers.

In our pilot study, 59 patients and 20 healthy volunteers were asked about the level of daily alcohol consumption, however without any statement success. Nevertheless, mean CDT in overall group was 1.5 (±0.9). Six (10.2%) patients had CDT over 2.5, and 40 (6.7%) over 1.2. The results obtained were taken into account by clinicians regarding perspective of further general treatment after conversation with the patients asking for lowering alcohol consumption.

In our study a statistical positive correlation was found between ALP, GGT (a marker for excessive alcohol consumption) with transferrin and possible correlation of NT pro-BNP with transferrin as shown on scatter plot (Figure 2). No statistical correlation between transferrin and troponin T was found. Determination of GGT and ALP concentrations increases the diagnostic sensitivity of liver damage and thus disorders of hepatocyte integrity. Alcohol damage to the liver is associated with the toxic effects of acetaldehyde and the development of reduction stress caused by an excess of reduced form of nicotinamide dinucleotide phosphate (NADPH). The consequence is disturbances in the oxidation processes in the liver cells leading to changes in the metabolism of carbohydrates, lipids, uric acid, and porphyrins. Identification of alcohol consumers based on family and community history, as well as the use of questionnaires might be optimized when specific diagnosis of alcohol abuse is taking place consequently. Desialylated transferrin as a marker of chronic alcohol consumption of high prognostic value enables the identification of a group of non-compliant patients, thus contributing to the effectiveness and optimization of treatment.

The results of ROC analyses for NT pro-BNP and troponin T were shown no correlation between marker of alcohol abuse and specific markers of heart failure tested during the study. NT pro-BNP as a one of the predictors of higher mortality in the course of heart failure with troponin T—a protein involved in the regulation of myocardial contraction are those almost routinely determined during the diagnosis of myocardial infarction, acute coronary syndromes, and prognosis of patients with other causes of heart damage [27,28,29]. Patients hospitalized due to cardiovascular diseases, including heart failure, often do not report or underestimate their excessive alcohol consumption in clinical interviews. As a result of patient untruthfulness regarding alcohol abuse, disorders described above, especially when NT pro-BNP and troponin T are increased, might easily progress and result in the patient's non-compliance with medical recommendations, such as development of irreversible consequences such as heart failure and/or qualification for heart transplantation or mechanical heart support.

Unlike other alcohol markers (such as GGT or mean red blood cell volume), desialylated transferrin levels do not increase in the presence of other diseases such as nonalcoholic liver disease. The CDT methodology has so far been validated by a reference laboratory and has shown satisfactory linearity and precision [30], and low false-positive rates are observed [31,32]. In our study, we have confirmed that CDT evaluation can be very informative for clinicians, as some patients are rather afraid to make a statement about some level of alcohol abuse.

The original methodology developed in the course of the pilot study has become a reliable analytical and applicable tool for the diagnosis and monitoring of patients with alcohol problems treated at the National Institute of Cardiology. An effective lifestyle modification via lowering or excluding alcohol consumption in patients with CVD could improve the quality of life, treatment effectiveness, and reduce possible adverse complications. The determination of the content of desialylated transferrin in blood serum may provide valuable support for the overall diagnosis and accurate monitoring of excessive alcohol consumption which might lead to chronic damage to the heart muscle. So far, CDT as marker of excessive level of alcohol consumption or complete abstinence is widely accepted for CVD patients. There are still many difficulties regarding the range of objective evaluation of the acceptable consumption of alcohol in individuals with increased cardiovascular risk and patients with cardiovascular diseases. The application of an accessible method to verify patients’ declarations of alcohol consumption may contribute to the development of more precise recommendations on alcohol consumption or abstinence in this group [4].

The use of a desialylated transferrin as a marker of alcohol consumption might improve the effectiveness of treatment and also identify those who need special care regarding abuse. More large-scale studies are needed to further confirm the diagnostic utility of carbohydrate-deficient transferrin.