Background: There is need for a reference method for transferrin glycoforms in serum to which routine immunologic methods for the alcohol marker carbohydratedeficient transferrin (CDT) can be traceable. We describe an improved HPLC method for transferrin glycoforms. Methods: Transferrin was iron-saturated by mixing the serum with ferric nitrilotriacetic acid, and lipoproteins were precipitated with dextran sulfate and calcium chloride. Separation of glycoforms was performed on a SOURCE 15Q anion-exchange column using salt gradient elution. Quantification relied on selective absorbance of the iron-transferrin complex at 470 nm. The relative amount of each glycoform was calculated as a percentage of the area under the curve, using baseline integration. Results: The HPLC system provided reproducible separation and quantification of the asialo-, monosialo-, disialo-, trisialo-, tetrasialo-, pentasialo-, and hexasialotransferrin glycoforms. Most importantly, disialo-and trisialotransferrin were almost baseline separated. The intra-and interassay CV for disialotransferrin were <5%. Serum and the pretreated samples were stable for at least 2 days at 22 or 4°C. Sera from 132 healthy controls contained [mean (SD)] 1.16 (0.25)% disialotransferrin, 4.77 (1.36)% trisialotransferrin, 80.18 (2.01)% tetrasialotransferrin, and 13.88 (1.69)% pentasialo-؉ hexasialotransferrin. In some cases of a high (>6%) trisialotransferrin, monosialotransferrin was detected at <0.25%. Asialotransferrin was not detected in control sera, but was detected in 57% of chronic heavy drinkers and in 62% of sera with >2% disialotransferrin.

Background The aim of this study was to compare the results of Phosphatidylethanol (PEth) and carbohydrate‐deficient transferrin (CDT) in blood as biomarkers of alcohol consumption in a large clinical cohort and to evaluate concentrations in relation to age and sex. Methods Results of PEth 16:0/18:1 in blood and CDT in serum were included, together with information of age and sex, which were extracted from a clinical chemistry database containing samples mostly from patients of primary care physicians and social care institutions. PEth concentrations were determined using Ultra Performance Convergence chromatography mass spectrometer. CDT was quantified by electrophoretic Capillary System. CDT values ≥ 1.7 %‐units and PEth values ≥ 0.31 µmol/L were considered to indicate heavy alcohol consumption. Results Samples from 6705 patients were included. The median age was 54.5 years, and 34 % were females. Only 47 % of the patients with PEth ≥ 0.31 µmol/L had increased CDT ≥ 1.7 %‐units examined in the same specimen (Cohen’s kappa was 0.43, p < 0.001). Patients above 50 years had significantly higher concentrations for both CDT (1.0 %‐units vs. 0.9 %‐units, p < 0.001) and PEth (0.340 µmol/L vs. 0.200 µmol/L, p < 0.001) compared with younger patients. Concentrations of CDT were significantly higher in males compared with females (p = 0.002), while no significant sex differences were seen for PEth (p = 0.465). Conclusions A high fraction of the patients had PEth values above the suggested cutoff for heavy drinking and normal CDT values, verifying the superior sensitivity of PEth compared with CDT. The effect of age seems to be minor for both markers. Higher concentrations of CDT, but not PEth, were seen in males, indicating that PEth, as opposed to CDT, might be formed equally in men and women. Therefore, the bias due to sex is possibly present only for CDT, not for PEth.

Background The aim of this study was to evaluate the quantitative relation between common clinical chemical analyses and ethanol use, measured by a combination of the two alcohol markers phosphatidylethanol (PEth) and carbohydrate‐deficient transferrin (CDT). Methods Results of PEth and CDT in whole blood and serum, respectively, were included, together with information on 10 different commonly measured clinical chemical analytes, as well as age and sex. PEth was analysed by UPC2‐MS/MS and CDT was measured by capillary electrophoresis. Results Samples from 4873 patients were included. The strongest relation to alcohol consumption as measured by PEth, when correcting for age and sex, was found for HDL‐C (standardized β = 0.472, p < 0.001), AST (standardized β = 0.372, p < 0.001), ferritin (standardized β = 0.332, p < 0.001) and GGT (standardized β = 0.325, p < 0.001). The relation to PEth was weak for total cholesterol, TG and ALP. No relation was found for Hb and LDL‐C. Conclusions When using PEth as a marker for alcohol consumption, this study demonstrated the quantitative relation to commonly used test as AST or GGT, but also an important relation to ferritin or HDL‐C. In clinical practice, elevated levels of these clinical chemical analytes should initiate further work‐up on possibly harmful alcohol use.

Primary biliary cirrhosis (PBC) is one of the few non-alcohol induced liver pathologies which causes false positive results in the evaluation of carbohydrate-deficient transferrin (CDT) for the diagnosis of alcohol misuse. This phenomenon has only been observed when using the CDTect assay (Pharmacia & Upjohn, Uppsala, Sweden). In this study, we evaluated CDT in female PBC patients (n = 14) by a new CDT procedure, the %CDT turbidimetric immunoassay (TIA, Axis Biochemicals, Oslo, Norway) using the isoelectric focusing/immunoblotting/laser densitometry (IEF/IB/LD, Specialty Laboratories, Santa Monica, CA, USA) procedure as the gold standard. One of the PBC patients tested CDT+ by IEF/IB/LD (cut-off >9 densitometry units, DU) and %CDT TIA (cut off >6%); one patient tested at the cut-off point of the IEF/IB/LD and another one tested at the cut-off point of the %CDT TIA. Thus, unlike CDTect, the %CDT TIA is a procedure that produces few false positives in PBC.

Background: The concentration of vitamin K1 in serum or plasma is the most common index for assessing vitamin K status. The aim of this study was to develop and validate a rapid and reliable routine method for quantifying vitamin K1 above 0.1 ng/mL. Semi-automation of a simple sample preparation with fast analysis by supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) was exploited. Methods: Vitamin K1 was extracted from 250-μL serum samples by the use of protein precipitation and reversedphase solid-phase extraction (SPE) in 96-well plates and quantified by SFC on a 2.1 × 100 mm Torus 1-Aminoanthracene (1-AA) column in 3.8 min with electrospray ionization-tandem mass spectrometry (MS/MS) detection. Results: This method shows good linearity in the concentration range of 0.1-50 ng/mL with a correlation coefficient of R 2 >0.999. Imprecision was satisfactory, with repeatability and reproducibility <10% CV. The lower limit of the measuring interval was 0.1 ng/mL, and no systematic bias was observed for the method, which used vitamin K1-d 7 as internal standard. Recovery of vitamin K1 in external quality controls was satisfactory compared to other laboratories participating in the external quality assurance scheme. The method is currently in routine use for analysis of serum samples. Conclusions: The method allows high-throughput reliable determination of vitamin K1 in serum in the range 0.1-50 ng/mL. IMPACT STATEMENT With the developed method, measurement of vitamin K1 level in patient serum samples can be performed rapidly and reliably. The method includes a semi-automated solid-phase extraction sample preparation method without the need of an evaporation step before quantification by supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS). The method provides a faster analysis than that of LC-MS/MS, and in a period of 3 months, the method has been applied to analysis of >5000 samples. The method is suitable for use in highthroughput medical laboratories.