Improved HPLC Method for Carbohydrate-deficient Transferrin in Serum

Abstract

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.

Toward standardization of carbohydrate-deficient transferrin (CDT) measurements: III. Performance of native serum and serum spiked with disialotransferrin proves that harmonization of CDT assays is possible

“…Three capillary zone electrophoresis (CE) methods were included: the CEofix CDT procedure [ 11 ] (Suarl é e, Belgium) and the Capillarys [ 12 ] and Minicap methods of Sebia (Lisses, France). Four HPLC methods were included: the candidate reference method [ 8 ], the %CDT by HPLC assay of Bio-Rad (Munich, Germany) with both standard [ 13 ] and fast procedures, and the ClinRep CDT [ 14 ] of Recipe (Munich, Germany). The immunonephelometric N Latex CDT assay [ 6 ] of Siemens (Marburg, Germany) was also included.…”

“…The CDT level in these pools was determined using the HPLC candidate reference method [ 8 ] and the relative concentrations of disialotransferrin were found to be approximately 1.2% for P1 and approximately 8.0% for P6. Then, four intermediate CDT levels (P2 -P5) were produced by a mathematical dilution (equal intervals) of P1 and P6 to obtain disialotransferrin concentrations of 2.6%, 3.9%, 5.3% and 6.6%.…”

“…This prompted the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) to initiate a Working Group aiming for standardization of CDT measurements (WG-CDT). In its first publication [ 7 ], disialotransferrin (disialylated monoglycan transferrin) was defined as the measurand for a reference method and the target analyte for the standardization work, and high-performance liquid chromatography (HPLC) [ 8 ] with photometric detection was proposed as the candidate reference method. A network of reference laboratories running this method was formed demonstrating good intra-and inter-laboratory performance [ 9 ].…”

Two Argentinean Siblings with CDG-Ix: A Novel Type of Congenital Disorder of Glycosylation?

“…High-performance liquid chromatography of transferrin (Tf-HPLC) analysis was based on the method described by Helander et al (2003).The HPLC system consisted of an Agilent 1100 Series liquid chromatography. Separation of the transferrin glycoforms was performed on a SOURCE 15Q PE 4.6/100 anion-exchange chromatography column (GE Healthcare) at 25 C by linear salt gradient elution at a flow rate of 1.0 ml/min.…”

“…Quantification of the transferrin glycoforms relied on the selective absorbance of the irontransferrin complex at 470 nm. The relative amount of each transferrin isoform was expressed as a percentage of the area under the curve (%AUC) (Helander et al 2003;Quintana et al 2009). …”