Improved HPLC method for carbohydrate-deficient transferrin in serum
(2003) In Clinical Chemistry 49(11). p.1881-1890- Abstract
- 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... (More)
- 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. (Less)
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https://lup.lub.lu.se/record/899827
- author
- Helander, A ; Husa, A and Jeppsson, Jan-Olof LU
- organization
- publishing date
- 2003
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Clinical Chemistry
- volume
- 49
- issue
- 11
- pages
- 1881 - 1890
- publisher
- American Association for Clinical Chemistry
- external identifiers
-
- pmid:14578320
- wos:000186157300012
- scopus:0242267940
- ISSN
- 0009-9147
- DOI
- 10.1373/clinchem.2003.023341
- language
- English
- LU publication?
- yes
- id
- 0b11ba3d-ddbb-4cdb-bee9-b0d4db84a23c (old id 899827)
- date added to LUP
- 2016-04-01 12:05:51
- date last changed
- 2022-03-20 23:28:21
@article{0b11ba3d-ddbb-4cdb-bee9-b0d4db84a23c,
abstract = {{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.}},
author = {{Helander, A and Husa, A and Jeppsson, Jan-Olof}},
issn = {{0009-9147}},
language = {{eng}},
number = {{11}},
pages = {{1881--1890}},
publisher = {{American Association for Clinical Chemistry}},
series = {{Clinical Chemistry}},
title = {{Improved HPLC method for carbohydrate-deficient transferrin in serum}},
url = {{http://dx.doi.org/10.1373/clinchem.2003.023341}},
doi = {{10.1373/clinchem.2003.023341}},
volume = {{49}},
year = {{2003}},
}