Improved HPLC method for carbohydrate-deficient transferrin in serum

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Improved HPLC method for carbohydrate-deficient transferrin in serum. / Helander, A; Husa, A; Jeppsson, Jan-Olof.

I: Clinical Chemistry, Vol. 49, Nr. 11, 2003, s. 1881-1890.

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Helander, A ; Husa, A ; Jeppsson, Jan-Olof. / Improved HPLC method for carbohydrate-deficient transferrin in serum. I: Clinical Chemistry. 2003 ; Vol. 49, Nr. 11. s. 1881-1890.

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TY - JOUR

T1 - Improved HPLC method for carbohydrate-deficient transferrin in serum

AU - Helander, A

AU - Husa, A

AU - Jeppsson, Jan-Olof

PY - 2003

Y1 - 2003

N2 - Background: There is need for a reference method for transferrin glycoforms in serum to which routine immunologic methods for the alcohol marker carbohydrate-deficient 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 run. 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 degreesC. 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 greater than or equal to2% disialotransferrin. Conclusions: The HPLC method fulfills the requirements of a preliminary reference method for CDT and should work for any combination of serum transferrin glycoforms. This method could also be useful for confirming positive CDT results by immunoassays in medico-legal cases. (C) 2003 American Association for Clinical Chemistry.

AB - Background: There is need for a reference method for transferrin glycoforms in serum to which routine immunologic methods for the alcohol marker carbohydrate-deficient 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 run. 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 degreesC. 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 greater than or equal to2% disialotransferrin. Conclusions: The HPLC method fulfills the requirements of a preliminary reference method for CDT and should work for any combination of serum transferrin glycoforms. This method could also be useful for confirming positive CDT results by immunoassays in medico-legal cases. (C) 2003 American Association for Clinical Chemistry.

U2 - 10.1373/clinchem.2003.023341

DO - 10.1373/clinchem.2003.023341

M3 - Article

VL - 49

SP - 1881

EP - 1890

JO - Clinical Chemistry

JF - Clinical Chemistry

SN - 0009-9147

IS - 11

ER -