Improved HPLC method for carbohydrate-deficient transferrin in serum.
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 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 degrees 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.


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.
Helander
Anders
A
Department of Clinical Neuroscience, Karolinska Institutet and Hospital, SE-171 76 Stockholm, Sweden. anders.helander@cns.ki.se
Husa
Asgeir
A
Jeppsson
Jan-Olof
JO
D000328:Adult; D000368:Aged; D002851:Chromatography, High Pressure Liquid; D005260:Female; D006031:Glycosylation; D006801:Humans; D008297:Male; D008875:Middle Aged; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D010783:Photometry; D020033:Protein Isoforms / Q000097:blood; D012015:Reference Standards; D012016:Reference Values; D014168:Transferrin / Q000031:analogs & derivatives* / Q000032:analysis*
D020033:Protein Isoforms; D014168:Transferrin; C056759:carbohydrate-deficient transferrin; D019158:N-Acetylneuraminic Acid