Carbohydrate-deficient Transferrin Research Articles

Terbium chelation, a specific fluorescent tagging of human transferrin. Optimization of conditions in view of its application to the HPLC analysis of carbohydrate-deficient transferrin (CDT).

Transferrin (Tf) is the major iron-transporting protein in the human body and, for this reason, has been extensively studied in biomedicine. This protein undergoes a complex glycosylation process leading to several glycoforms, some of which are important in the diagnosis of alcohol abuse and of congenital glycosylation defects under the collective name of carbohydrate-deficient transferrin (CDT). Exploiting the Tf ability to bind not only iron but also other ions, specific attention has been devoted to binding activity towards Tb3+, which was reported to greatly enhance its intrinsic fluorescence upon the interaction with Tf. However, the structural properties of the Tb3+-Tf complex have not been described so far. In the present work, the formation of the Tf-Tb3+ complex has been investigated by the employment of several biophysical techniques, such as fluorescence resonance energy transfer (FRET), "native" mass spectrometry (MS), and near-UV circular dichroism (CD). Each method allowed the detection of the Tf-Tb3+ complex, yielding a specific signature. The interaction of Tb3+ with Fe3+-free Tf (apoTf) has been described in terms of stoichiometry, affinity, and structural effects in comparison with Fe3+. These experiments led to the first direct detection of the Tf-Tb3+ complex by MS, indicating a 1:2 stoichiometry and allowing the investigation of structural effects of metal binding. Either Tb3+ or Fe3+ binding affected protein conformation, inducing structural compaction to a similar extent. Nevertheless, near-UV CD and pH-dependence profiles suggested subtle differences in the coordination of the two metals by Tf side chains. Experimental conditions that promote complex formation have been identified, highlighting the importance of alkaline pH and synergistic ions, such as carbonate. On the basis of these studies, sample pretreatment, separation, and detection conditions of a high-performance liquid chromatographic method for CDT analysis are optimized, achieving relevant increase (by a factor of ∼3) of analytical sensitivity. Graphical abstract Schematic representation of HPLC-separated transferrin glycoforms detected by fluorescence emission of the terbium ions bound to the protein.

Identifying chronic heavy alcohol use in emergency general surgery patients: a pilot study

BackgroundChronic heavy alcohol (CHA) use has been associated with perioperative complications. Emergency general surgery (EGS) patients are not routinely screened for CHA. If screened, it is usually for hazardous use...

Why measure carbohydrate deficient transferrin?

The acute or chronic harmful use of alcohol is among the top five risk factors for disease, disability and death worldwide.1 Monitoring alcohol consumption is an important aspect of the...

Analysis of genetic variants of transferrin in human serum after desialylation by capillary zone electrophoresis and capillary isoelectric focusing.

Capillary electrophoresis analysis of transferrin in human serum is used to assess genetic variants after desialylation with neuraminidase and iron saturation to reduce the complexity of the transferrin pattern and thus facilitate the recognition of transferrin polymorphisms. Asialo-transferrin forms are analyzed by capillary zone electrophoresis using assay conditions as for the monitoring of carbohydrate-deficient transferrin or by capillary isoelectric focusing in a pH 5-8 gradient which requires immunoextraction of transferrin prior to analysis. With the carrier ampholytes used, peaks for iron saturated and iron depleted transferrin are monitored which indicates complexation of iron ions by carrier ampholytes. For BC, CD, and BD genetic variants, the expected peaks for B, C, and D forms of transferrin were detected with both methods. Monitoring of CC patterns revealed three cases, namely those producing double peaks in both methods, a double peak in capillary isoelectric focusing only and a double peak in capillary zone electrophoresis only. For all samples analyzed, data obtained by capillary isoelectric focusing could be confirmed with gel isoelectric focusing. The two capillary electrophoresis methods are shown to represent effective tools to assess unusual transferrin patterns, including genetic variants with dissimilar abundances of the two forms.

Improved detection of alcohol consumption using the novel marker phosphatidylethanol in the transplant setting: results of a prospective study

Phosphatidylethanol (PEth) is a new, highly specific alcohol marker. The aim of this study was to assess its diagnostic value in the liver transplant setting. In 51 pre- and 61 post-transplant patients with underlying alcoholic liver disease PEth, ethanol, methanol, carbohydrate-deficient transferrin (CDT), and ethyl glucuronide in urine (uEtG) and hair (hEtG) were tested and compared with patients' questionnaire reports. Twenty-eight (25%) patients tested positive for at least one alcohol marker. PEth alone revealed alcohol consumption in 18% of patients. With respect to detection of alcohol intake in the preceding week, PEth showed a 100% sensitivity. PEth testing was more sensitive than the determination of ethanol, methanol, CDT or uEtG alone [sensitivity 25% (confidence interval (CI) 95%, 7-52%), 25% (7-52%), 21% (6-45%) and 71% (41-91%), respectively], or ethanol, methanol and uEtG taken in combination with 73% (45-92%). Specificity of all markers was 92% or higher. Additional testing of hEtG revealed alcohol consumption in seven patients, not being positive for any other marker. Phosphatidylethanol was a highly specific and sensitive marker for detection of recent alcohol consumption in pre- and post-transplant patients. The additional determination of hEtG was useful in disclosing alcohol consumption 3-6 months retrospectively.

How strong is the evidence for using blood biomarkers alone to screen for alcohol consumption during pregnancy? A systematic review

Accurate and early identification of women at risk from alcohol consumption during pregnancy allows education and support programmes to be targeted at those most in need. We aimed to conduct a systematic review to compare the efficacy of blood analysis and maternal self-report in detecting at risk women during pregnancy. This review investigated diagnostic accuracy. We searched four databases (Medline, Embase, Psychinfo and CINAHL) for relevant articles and conducted hand searches of recent issues of key journals in the field. No restriction was placed on inclusion in terms of publication date or language. Studies were deemed eligible if they were original research and included a direct comparison of the results of blood biomarker analysis and self-reported alcohol use for the detection of alcohol consumption in pregnant women. Quality appraisal of included studies was conducted using the QUADAS II tool. Eight studies met the inclusion criteria. Gamma-glutamyltransferase (GGT) was investigated in five studies, mean corpuscular volume (MCV) and phosphatidylethanol (PEth) in three studies and carbohydrate deficient transferrin (CDT), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and whole blood associated acetaldehyde assay (WBAA) were each investigated in two studies. Although all of the studies were rated of good methodological quality, none of the biomarkers had both high sensitivity and specificity when compared to self-report. There was some evidence that a combination of biomarkers, or combining biomarkers with self-report, increases accuracy. In summary, the blood biomarkers examined were of limited use in screening for low and moderate alcohol consumption in pregnancy when compared to self-report. However, certain biomarkers, such and CDT and PEth may complement self-report and help improve the accuracy of diagnosis.

Reprint of Standardisation and use of the alcohol biomarker carbohydrate-deficient transferrin (CDT)

Carbohydrate-deficient transferrin (CDT) is a glycoform profile of serum transferrin that increases in response to sustained high alcohol intake and over the last decades has become an important alcohol biomarker with clinical and forensic applications. However, the wide range of CDT measurement procedures has resulted in lack of uniform results and reference limits, and hampered comparison of results. In 2005, the IFCC therefore founded a special working group (WG) aiming for standardisation of CDT measurement. This review summarises the history of CDT and the actions taken by the WG-CDT. Initial steps included the definition of the measurand (serum disialotransferrin to total transferrin fraction expressed in %), and the determination of a well-defined anion-exchange HPLC procedure as the candidate reference measurement procedure (cRMP). Subsequent achievements were the establishment of a network of reference laboratories to perform the cRMP, setting a reference interval, and development of a reference material based on human serum for which the laboratory network assign values. Using a set of reference materials for calibration allowed for achieving equivalence of results of all present CDT measurement procedures. The final steps of the WG-CDT have been a full validation of the cRMP to make it an IFCC approved RMP, and providing guidance for international standardisation of all CDT measurement procedures.

Comparison of transferrin isoform analysis by capillary electrophoresis and HPLC for screening congenital disorders of glycosylation.

Transferrin, a major glycoprotein has different isoforms depending on the number of sialic acid residues present on its oligosaccharide chain. Genetic variants of transferrin as well as the primary (CDG) & secondary glycosylation defects lead to an altered transferrin pattern. Isoform analysis methods are based on charge/mass variations. We aimed to compare the performance of commercially available capillary electrophoresis CDT kit for diagnosing congenital disorders of glycosylation with our in-house optimized HPLC method for transferrin isoform analysis. The isoform pattern of 30 healthy controls & 50 CDG-suspected patients was determined by CE using a Carbohydrate-Deficient Transferrin kit. The results were compared with in-house HPLC-based assay for transferrin isoforms. Transferrin isoform pattern for healthy individuals showed a predominant tetrasialo transferrin fraction followed by pentasialo, trisialo, and disialotransferrin. Two of 50 CDG-suspected patients showed the presence of asialylated isoforms. The results were comparable with isoform pattern obtained by HPLC. The commercial controls showed a <20% CV for each isoform. Bland Altman plot showed the difference plot to be within +1.96 with no systemic bias in the test results by HPLC & CE. The CE method is rapid, reproducible and comparable with HPLC and can be used for screening Glycosylation defects.

Carbohydrate-Deficient Transferrin as a Biomarker for Screening At-Risk Drinking in Elderly Men.

BackgroundCarbohydrate-deficient transferrin (CDT) is a useful biomarker to identify excessive alcohol consumption; however, few studies have validated the %CDT cut-off value in elderly men. This study estimated the optimal %CDT cut-off value that could identify excessive alcohol consumption in men aged ≥65 years.MethodsThis retrospective study included 120 men who visited the department of family medicine at Chungnam National University Hospital for health check-up between January 2010 and August 2013. At-risk drinking included heavy- and binge drinking. Heavy drinking was defined as more than seven standard drinks/wk, and binge drinking was defined as more than three standard drinks/d. The cut-off %CDT values for at-risk drinking were determined using receiver operating characteristic (ROC) curves.ResultsBased on the ROC curves, the optimal %CDT cut-off values in ≥65-year-old men were 1.95% for at-risk drinking, 1.81% for heavy drinking, and 2.07% for binge drinking. The sensitivity, specificity, and positive and negative predictive values were 58.7%, 83.6%, 69.2%, and 76.2% for at-risk drinking, respectively. The AUROC were >0.7 for all three evaluated cut-offs.ConclusionOur results suggest that the %CDT cut-off value for at-risk drinking in elderly Korean men (≥65 years) should be readjusted to a lower value of 1.95%.

IFCC approved HPLC reference measurement procedure for the alcohol consumption biomarker carbohydrate-deficient transferrin (CDT): Its validation and use

Carbohydrate-deficient transferrin (CDT) is used as a biomarker of sustained high alcohol consumption. The currently available measurement procedures for CDT are based on various analytical techniques (HPLC, capillary electrophoresis, nephelometry), some differing in the definition of the analyte and using different reference intervals and cut-off values. The Working Group on Standardization of CDT (WG-CDT), initiated by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), has validated an HPLC candidate reference measurement procedure (cRMP) for CDT (% disialotransferrin to total transferrin based on peak areas), demonstrating that it is suitable as a reference measurement procedure (RMP) for CDT. Presented is a detailed description of the cRMP and its calibration. Practical aspects on how to treat genetic variant and so-called di-tri bridge samples are described. Results of method performance characteristics, as demanded by ISO 15189 and ISO 15193, are given, as well as the reference interval and measurement uncertainty and how to deal with that in routine use. The correlation of the cRMP with commercial CDT procedures and the performance of the cRMP in a network of laboratories are also presented. The performance of the CDT cRMP in combination with previously developed commutable calibrators allows for standardization of the currently available commercial measurement procedures for CDT. The cRMP has recently been approved by the IFCC and will be from now on be known as the IFCC-RMP for CDT, while CDT results standardized according to this RMP should be indicated as CDTIFCC.

Fluorescent adduct formation with terbium: a novel strategy for transferrin glycoform identification in human body fluids and carbohydrate-deficient transferrin HPLC method validation.

This paper puts forward a new method for the transferrin (Tf) glycoform analysis in body fluids that involves the formation of a transferrin-terbium fluorescent adduct (TfFluo). The key idea is to validate the analytical procedure for carbohydrate-deficient transferrin (CDT), a traditional biochemical serum marker to identify chronic alcohol abuse. Terbium added to a human body-fluid sample produced TfFluo. Anion exchange HPLC technique, with fluorescence detection (λ exc 298nm and λ em 550nm), permitted clear separation and identification of Tf glycoform peaks without any interfering signals, allowing selective Tf sialoforms analysis in human serum and body fluids (cadaveric blood, cerebrospinal fluid, and dried blood spots) hampered for routine test. Serum samples (n = 78) were analyzed by both traditional absorbance (Abs) and fluorescence (Fl) HPLC methods and CDT% levels demonstrated a significant correlation (p < 0.001 Pearson). Intra- and inter-runs CV% was 3.1 and 4.6%, respectively. The cut-off of 1.9 CDT%, related to the HPLC Abs proposed as the reference method, by interpolation in the correlation curve with the present method demonstrated a 1.3 CDT% cut-off. Method comparison by Passing-Bablok and Bland-Altman tests demonstrated Fl versus Abs agreement. In conclusion, the novel method is a reliable test for CDT% analysis and provides a substantial analytical improvement offering important advantages in terms of types of body fluid analysis. Its sensitivity and absence of interferences extend clinical applications being reliable for CDT assay on body fluids usually not suitable for routine test. Graphical Abstract The formation of a transferrin-terbium fluorescent adduct can be used to analyze the transferrin glycoforms. The HPLC method for carbohydrate-deficient transferrin (CDT%) measurement was validated and employed to determine the levels in different body fluids.

Infrared analysis of lipoproteins in the detection of alcohol biomarkers.

Alcoholism is a major public health problem. Alcohol causes modifications in the composition and concentration of lipoproteins and influences the enzymes and transfer proteins that transform lipoproteins in plasma. Alcohol is associated with the presence of alcohol biomarkers (fatty acid ethyl esters [FAEEs] and phosphatidylethanol [PEth]) in lipoproteins. We explore the possibilities of detecting alcohol biomarkers in non-high-density-lipoproteins (non-HDLs) precipitated from serum using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). Analyzes were carried out on stored serum samples, with known % carbohydrate-deficient transferrin (CDT) values, included in a driver's license regranting program under the control of the Belgian Institute of Road Safety. The study consisted of 127 control samples (CDT≤1.3%) and 114 alcoholic samples (CDT>1.3%). Liver enzymes, CRP, triglycerides, total, HDL- and LDL-cholesterol values were determined. Non-HDLs were precipitated with sodium phosphotungstate and MgCl2 and analyzed using ATR-FTIR in the range from 4500 cm-1 to 450 cm-1 using a Perkin Elmer ATR-FTIR Spectrometer Two. The area under the curve of the 1130-990 cm-1 region (AUC1130-990 cm-1) was able to discriminate controls from alcoholics (p<0.0001) due to the presence of FAEEs in lipoproteins. Multiple regression analysis significantly predicted the AUC1130-990 cm-1 (adj. r2=0.13, p<0.0001). Significant correlations were found between AUC1130-990 cm-1 and CDT values (r=0.32, p<0.0001), AST/ALT ratio (r=0.21, p=0.001). GGT showed no significant correlation. Infrared analysis of lipoproteins is a potential tool in the detection of alcohol biomarkers.

Relationship of Abnormal Chromatographic Pattern for Carbohydrate-Deficient Transferrin with Severe Liver Disease

Serum carbohydrate-deficient transferrin (CDT) is a validated test for chronic heavy alcohol drinking, but CDT abnormalities have been associated with liver disease, limiting its use in these patients. We report here on the association between poor chromatographic resolution of disialotransferrin from trisialotransferrin (the so-called 'di-tri bridging') and liver disease severity and etiology. Subjects were patients in whom detailed clinical data, including histology results, were available on their existing liver diseases (n=139). Percent disialo-CDT (%dCDT) was measured by high-performance liquid chromatography, and the risks for di-tri bridging associated with cirrhosis, with and without adjustment for alcohol use and alcohol-related liver disease, were estimated. Di-tri bridging was present in 22/73 (30%) cirrhotic subjects and 7/66 (11%) non-cirrhotic subjects. The unadjusted risk for di-tri bridging in cirrhotics relative to non-cirrhotics was 3.6 (95%confidence interval 1.4-9.2). Adjustment for alcohol-related liver disease and current drinking had little effect on this estimate (adjustedodds ratio 3.4), and neither alcohol-related liver disease nor current drinking were independently associated with di-tri bridging after accounting for the effect of cirrhosis. The presence of di-tri bridging was associated with cirrhosis in individuals with both alcohol-related and non-alcoholic liver disease, although most cirrhotic subjects did not exhibit di-tri bridging. When di-tri bridging is seen in individuals being tested for chronic heavy drinking, investigation for cirrhosis should be considered. There are known liver-disease-associated abnormalities in CDT. In this study, we found that such abnormalities were strongly associated with cirrhosis rather than less-advanced disease, but were only clinically evident in 30% of cirrhotics. Abnormalities also occurred in severe hepatitis without cirrhosis and were not specific for liver disease etiology.

Assays of Gamma-Glutamyl Transferase and Carbohydrate-Deficient Transferrin Combination from Maternal Serum Improve the Detection of Prenatal Alcohol Exposure.

Alcohol use during pregnancy leads to detrimental effects on fetal development. As self-reports by mothers are known to be unreliable for assessing prenatal alcohol exposure, there is a need for sensitive and specific biomarkers for identifying those at risk for alcohol-affected offspring. We measured serum gamma-glutamyl transferase (GGT), carbohydrate-deficient transferrin (CDT), a mathematically formulated combination of GGT and CDT (GGT-CDT), and ethylglucuronide (EtG) concentrations from 1,936 mothers with a positive (n=480) or negative (n=1,456) history of alcohol use at the time of pregnancy. The material included 385 alcohol-abusing mothers who subsequently gave birth to children with fetal alcohol syndrome (FAS) and 1,551 mothers without FAS children including 95 women who reported a median of 1.0 standard drinks of alcohol per day during pregnancy and 1,456 nondrinking controls. Among those without FAS outcome, there were 405 mothers with gestational diabetes mellitus (GDM) and 745 mothers representing lifelong abstainers. Mothers of FAS children had higher mean GGT, CDT, GGT-CDT, and EtG levels than abstainers (p<0.001 for all comparisons) or mothers reporting some alcohol consumption but whose children were not diagnosed with FAS (p<0.001 for all comparisons). In receiver operating characteristic analyses using cutoffs based on abstainers, the area under the curves (AUCs) for GGT-CDT (0.873) were higher than those of GGT (0.824), CDT (0.776), or EtG (0.584) for differentiating the mothers of FAS children and abstainers. Unlike CDT, this combination marker also differed significantly between drinking mothers without FAS outcome and abstainers (AUC=0.730, p<0.001). In comparisons adjusted for GDM and body mass index, the group of mothers who had reported a median of 1.0 standard drinks of alcohol per day during pregnancy also differed from the group reporting no current alcohol intake in GGT (p<0.02) and GGT-CDT (p<0.01) levels. Combination of GGT and CDT improves the identification of prenatal alcohol exposure and associated high-risk pregnancies. A more systematic use of biomarkers may help intervention efforts to prevent alcohol-induced adverse effects on fetal development.

Cross-sectional study on N,N-dimethylformamide (DMF); effects on liver and alcohol intolerance.

There are still concerns regarding occupational exposure to hepatotoxic DMF. This study was designed to evaluate possible liver damaging effects of DMF under current workplace conditions in synthetic fibres industries. Among other laboratory parameters, liver function parameters (alkaline phosphatase (ALP), aspartate aminotransferase, alanine aminotransferase and gamma-glutamyltransferase), the mean corpuscular erythrocyte volume (MCV) and carbohydrate-deficient transferrin (CDT) of the workforce of two companies present at the days of study were investigated. Internal exposure to DMF was assessed via three different biomarkers [sum of N-methylformamide and N-hydroxymethyl-N-methylformamide, N-acetyl-S-(N-carbamoyl)cysteine (AMCC) and 3-methyl-5-isopropylhydantoin (MIH)]. Alcohol consumption was assessed by means of direct ethanol metabolites (ethylglucuronide and ethylsulfate). None of the tested liver enzyme activities showed a positive association with any of the three exposure markers, nor did CDT and MCV. CDT was negatively associated with AMCC and the ALP activity negatively with all three exposure markers. Changes in liver function are seen mainly in conjunction with ethanol consumption but also with increasing body weight and age. MCV was associated with smoking. Almost half of the workers stated to experience alcohol flush reaction. The present study indicates that long-term exposure to DMF, which was specified by median urinary AMCC levels of 4.84mg/g creatinine and DMF haemoglobin adduct levels of 60.5nmol/MIH/g globin, respectively, does not result in any adverse liver effects. In contrast, these DMF exposure levels still elicit certain alcohol intolerance reactions.

Alcoholic cardiomyopathy : The result of dosage and individual predisposition.

The individual amount of alcohol consumed acutely or chronically decides on harm or benefit to a person’s health. Available data suggest that one to two drinks in men and one drink in women will benefit the cardiovascular system over time, one drink being 17.6 ml 100 % alcohol. Moderate drinking can reduce the incidence and mortality of coronary artery disease, heart failure, diabetes, ischemic and hemorrhagic stroke. More than this amount can lead to alcoholic cardiomyopathy, which is defined as alcohol toxicity to the heart muscle itself by ethanol and its metabolites. Historical examples of interest are the Munich beer heart and the Tübingen wine heart. Associated with chronic alcohol abuse but having different etiologies are beriberi heart disease (vitamin B1 deficiency) and cardiac cirrhosis as hyperdynamic cardiomyopathies, arsenic poising in the Manchester beer epidemic, and cobalt intoxication in Quebec beer drinker’s disease. Chronic heavy alcohol abuse will also increase blood pressure and cause a downregulation of the immune system that could lead to increased susceptibility to infections, which in turn could add to the development of heart failure. Myocardial tissue analysis resembles idiopathic cardiomyopathy or chronic myocarditis. In the diagnostic work-up of alcoholic cardiomyopathy, the confirmation of alcohol abuse by carbohydrate deficient transferrin (CDT) and increased liver enzymes, and the involvement of the heart by markers of heart failure (e.g., NT-proBNP) and of necrosis (e.g., troponins or CKMb) is mandatory. Treatment of alcoholic cardiomyopathy consists of alcohol abstinence and heart failure medication.

Quantification of EtG in hair, EtG and EtS in urine and PEth species in capillary dried blood spots to assess the alcohol consumption in driver’s licence regranting cases

BackgroundIn Belgium, the analysis of indirect biomarkers such as carbohydrate deficient transferrin (CDT%), gamma-glutamyltransferase (GGT), aspartate aminotransferase/alanine aminotransferase (AST/ALT) and mean corpuscular volume (MCV), is currently used to monitor the alcohol consumption in cases of fitness to drive assessment. We evaluated the use of direct ethanol markers for this purpose, exclusively determined in matrices obtained via non- or minimally invasive sampling. MethodsThree validated quantitative methods (ethylglucuronide (EtG) in hair and urine, ethylsulfate (EtS) in urine, and phosphatidylethanol species (PEth 16:0/18:1, PEth 18:1/18:1 and PEth 16:0/16:0) in capillary dried blood spots (C-DBS)) were used. Fifty volunteers, for whom fitness to drive had to be assessed and for whom a blood analysis for indirect biomarkers was requested, were included in the study. The sampling and analysis of hair, urine and C-DBS were added to the process currently used. ResultsHair EtG (24/50) and C-DBS PEths (29/50) are more sensitive than the currently used indirect biomarkers (13/50 for CDT%) to detect excessive and chronic alcohol consumption and allow to disprove an abstinence period. Urinary EtG and EtS are useful parameters to determine recent alcohol consumption. ConclusionThe combined use of the three strategies allows better inference about the evolution of the alcohol consumption prior to the sampling. Moreover, the exclusive use of non- or minimally invasive sampling (hair, urine and C-DBS) allows this to be performed directly during the fitness to drive assessment by regular staff members. This approach offers the potential to improve the Belgian driver’s licence regranting process.

Use of finger-prick dried blood spots (fpDBS) and capillary electrophoresis for carbohydrate deficient transferrin (CDT) screening in forensic toxicology.

Continued progress in chronic alcohol abuse investigation requires the development of less invasive procedures for screening purposes. The application of finger-prick and related dried blood spots (fpDBS) for carbohydrate deficient transferrin (CDT) detection appears suitable for this aim. Therefore, the goal of this project was to develop a screening method for CDT using fpDBS with CZE analysis. Blood samples prepared by finger-prick were placed on DBS cards and left to air dry; each dried fpDBS disc was shredded into small pieces and suspended in acid solution (60 μL of HCl 120 mmol/L). After centrifugation (10 min at 1500 × g), the collected sample was adjusted to pH 3.5. After an overnight incubation, the pH was neutralised and an iron rich solution was added. After 1 h, CZE analysis was carried out. A group of 47 individuals was studied. Parallel serum samples were collected from each investigated subject and the %CDT for each sample was measured using HPLC and CZE techniques. The fpDBS transferrin sialo isoform electropherograms were similar to those obtained with serum. Moreover, fpDBS CZE CDT percentage levels demonstrated significant statistical correlation with those obtained from serum for both HPLC and CZE %CDT (p < 0.01; r2 = 0.8913 and 0.8976, respectively), with %CDT from 0.8 to 13.7% for fpDBS and from 0.7 to 12.7% for serum. The newly developed fpDBS procedure for CDT analysis provides a simple and inexpensive tool for use in population screening.

Fetal Alcohol Syndrome and Maternal Alcohol Biomarkers in Sera: A Register-Based Case-Control Study.

Fetal alcohol syndrome (FAS) is a well-known consequence of prenatal alcohol exposure. However, women tend to deny or underreport their alcohol use during pregnancy. The aim of this study was to explore the usability of various alcohol biomarkers for FAS screening in a data set without information on self-reported alcohol use. A nationwide register study with a case-control design was conducted. The target population consisted of all live births in Finland from 1987 to 2005. FAS cases (n=565) were identified from the Finnish Register of Congenital Malformations. Mothers of FAS cases and their controls were selected in a ratio of 1 to 2 from the Finnish Maternity Cohort (FMC). Background information was obtained from the Finnish Medical Birth Register. Serum samples, collected at the mother's first visit to the maternity care, were obtained from the national FMC biobank. Biomarkers of alcohol consumption, gamma-glutamyltransferase (GGT), carbohydrate-deficient transferrin (%CDT), combination of GGT and CDT (GGT-CDT), and ethylglucuronide (EtG) were analyzed from mothers of FAS cases (n=385) and their controls (n=745). Median levels of all biomarkers were significantly higher among the mothers of FAS children than in control mothers. Using previously validated cutoffs for EtG, GGT, %CDT, and GGT-CDT, nearly half (46%) of the mothers with affected offspring could be identified. The predictive association was highest for GGT-CDT combination and significant also for all the other biomarkers. In this explorative case-control study, we demonstrate that the FMC biobank can be used to screen alcohol biomarkers for epidemiological research purposes. According to our results, the use of alcohol biomarkers during the first trimester may help to identify the high-risk pregnancies for FAS. A more systematic use of alcohol biomarkers at maternity care may open new possibilities for screening and intervention of alcohol use among pregnant mothers.

Standardisation and use of the alcohol biomarker carbohydrate-deficient transferrin (CDT)

Carbohydrate-deficient transferrin (CDT) is a glycoform profile of serum transferrin that increases in response to sustained high alcohol intake and over the last decades has become an important alcohol biomarker with clinical and forensic applications. However, the wide range of CDT measurement procedures has resulted in lack of uniform results and reference limits, and hampered comparison of results. In 2005, the IFCC therefore founded a special working group (WG) aiming for standardisation of CDT measurement. This review summarises the history of CDT and the actions taken by the WG-CDT. Initial steps included the definition of the measurand (serum disialotransferrin to total transferrin fraction expressed in %), and the determination of a well-defined anion-exchange HPLC procedure as the candidate reference measurement procedure (cRMP). Subsequent achievements were the establishment of a network of reference laboratories to perform the cRMP, setting a reference interval, and development of a reference material based on human serum for which the laboratory network assign values. Using a set of reference materials for calibration allowed for achieving equivalence of results of all present CDT measurement procedures. The final steps of the WG-CDT have been a full validation of the cRMP to make it an IFCC approved RMP, and providing guidance for international standardisation of all CDT measurement procedures.