THE HOUSE OF CHROMATOGRAPHY via C. Menotti… · Quantitationof Carbohydrate DeficientTransferrinin...
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Quantitation of Carbohydrate Deficient Transferrin in Serum: a New Method using Anion Exchange HPLC L. Lloyd 3 , R. Nilsson 1 , and A. Medin 2 1 Dept of Clinical Chemistry, Växjö Hospital, Sweden 2 Scantec Lab AB, Partille, Sweden 3 Polymer Laboratories, Abstract Prolonged, excessive consumption of alcohol is known to affect the glycosylation states of proteins. In Sweden the presence of elevated levels of carbohydrate deficient transferrin, CDT, is an accepted clinical marker for disproportionate alcohol consumption. There are seven possible isoforms of transferrin and the relative levels of these glycosylation states has been shown to be an indicator of excess alcohol consumption. We present an improved anion exchange HPLC method capable of resolving the isoforms of transferrin. Its potential suitability as an assay for the detection of alcohol abuse is demonstrated. Introduction High consumption of alcohol has several effects on the human body, which can be determined by laboratory analysis. Damage to cells can be estimated by the leakage of enzymes and is determined as serum levels of the liver enzymes glutamyl transferase (SGT), alanine amino transferase (SALAT) and aspartate amino transferase (SASAT). The enzyme levels are known to increase as a result of high alcohol intake over time and although alcohol is not the only factor which can result in an increase in the serum levels of these enzymes, alcohol is the most common factor, and, so their measurement is used in the treatment of alcoholism and alcohol related disease. However, these tests do not give the specificity needed for the determination of alcohol abuse and so an alternative assay which has improved specificity is required. A general effect on the glycosylation of proteins is observed with a high and prolonged exposure to alcohol and its metabolites. This effect is clearly seen with transferrin, an iron transport protein, which has carbohydrate side chains that have terminal sialic acid residues. There are seven possible isoforms of transferrin. In normal healthy individuals 80% of the protein is in its tetrasialo form, 14% in the penta and hexasialo forms, 5% in trisialo and 1% is present as disialo transferrin. Asialo transferrin is not detected in normal healthy subjects but is found in 50% of alcoholics. An increase in the levels of asialo, mono and disialo forms to over two percent has been correlated with a high daily intake of alcohol. The term carbohydrate deficient transferrin, CDT, relates to the total of these three forms. Carbohydrate deficient transferrin can be determined in plasma, PCDT, urine, U CDT, and serum, SCDT and in Sweden it is the blood serum level of carbohydrate deficient transferrin, SCDT, that is an accepted marker for extensive and prolonged consumption of alcohol 1 . These sialic acids are charged and thus confer differences to the net protein charge depending on the number of sialic acids that are present as part of the protein. As a consequence of the different net charge the transferrin glycosylation variants can be successfully resolved using anion exchange chromatography (IEC). Recently, an HPLC method utilising anion exchange chromatography to quantify the disialo form was reported 2 . This method gives a specificity of 99% but does not quantify the individual form of SCDT and has a relatively long cycle time. It was anticipated by Roland Nilsson, Klin Kem Lab Växjö Lasarett, that for an assay further improvements could be obtained by quantification of all the glycosylation states and reducing the assay cycle time – improving the through put. An improvement in the precision of the assay would be achieved if a gain in resolution could be obtained so that the individual forms could be quantified. The work presented here was carried out by Roland Nilsson and presents an assay for SCDT which meets these criteria. Växjö hospital where this work was carried out. HPLC System Column:PLSAX 1000Å 5 μm, 50 x 4.6 mm ID Eluent A: 20 mM BisTris, pH 6.4 Eluent B: 20 mM BisTris, pH 6.2 + 0.25 M NaCl Eluent C: 2 M NaCl Gradient: 0 min 6% B 1 min 13% B 4 min 24% B 6.5 min 37% B 6.6 min 38% B Cleaning: 2 min with 100% C Eqillibration: 4.4 min with 6% B Flow Rate: 1 mL/min Temperature: ambient Detection: UV, 460 nm Injection: 30 μL Run time: 9 min Cycle time: 13 min Sample Preparation Venous 10 mL aliquots of blood were collected in vacuum tubes, centrifuged for ten minutes at 12001400 g, and the supernatant decanted into new tubes. From this 100 μL of serum was transferred into a 1.5 mL micro centrifuge tube and lipoproteins were precipitated by the addition of 20 μL “FeNTA” reagent* and 500 μL H 2 O. Then 20 μL of precipitating reagent** was added with gentle mixing and the tubes were left at 4 ˚C overnight. The tubes were centrifuged at 20000 g for 10 minutes after which 150 μL of the clear supernatant was transferred into 300 μL (12 x 32 mm) plastic vials. *FeNTA reagent: 10 mM nitrilo acetic acid trisodium salt; FeCl 3 in H 2 O ** Precipitating reagent: 50% 1 M MgCl 2 solution in H 2 O / 50% 2% solution of Na dextran sulphate in H 2 O (v/v). Chromatography A 30 μL aliquot of the prepared serum sample was used for each assay. A salt and pH gradient was used for the separation of the transferrin isoforms, as detailed in the HPLC section. The run time was nine minutes and the total cycle time was 13 minutes. The glycosylation forms of the transferrin were separated based on the number of sialic acid residues; the more sialic acid residues the longer the retention time (Figure 1) shows the chromatogram of a sample from a healthy individual. Figure 1: Chromatogram showing the five sialo variants of transferrin in a ‘normal’ sample, di, tri, tetra, penta and hexasialo. Figure 2: Chromatogram showing the elution position of asialo transferrin. Figure 3: Chromatogram showing the elution position of monosialo transferrin. The anion exchange HPLC method is able to resolve the seven glycosylation forms of transferrin (Figures 1, 2 and 3). However, the asialo form is only seen in very rare cases and is of little diagnostic value as it occurs with elevated levels of the disialo form. The monosialo form also only occurs in very rare cases but is sufficiently well resolved to be identified during the assay. 0 min 12 tetra penta tri di hexa 0 min 12 high di-sialo a-sialo 0 min 12 mono-sialo Data Calculation and Interpretation The assay chromatograms are evaluated by assigning the five peaks correlating to the isoforms, di, tri, tetra, penta, and hex sialo forms and calculating the areas of each peak. The areas for the individual peaks are then combined to give a measure of the total transferrins present. The SCDT percent value is then calculated by dividing the area of the disialo form with the area for total transferrins. The normal result for SCDT in healthy adults with low or moderate alcohol consumption is 1% SCDT or lower (Figure 1). Values greater than 2% indicate high and continued intake of alcohol (Figure 4). Figure 4: Chromatogram showing an enhanced level of disialo transferrin as would be expected from an adult sample after prolonged high intake of alcohol. Results and Discussion This method was evaluated relative to the previously reported method using a regression study. Data from parallel analyses with both methods on routine patient investigations and analysis of control samples was also carried out over three months. A single PLSAX 1000Å 8μm column was used throughout. The previous method used a 100 mm x 4.6 mm ID Source15Q 4.6/100PE column packed with anion exchange material with a particle size of 15 μm. The regression analysis in Figure 5 shows excellent correlation with the older method, slope = 1.01 and r = 0.9967, for the 78 patient samples analyzed. The results spanned the complete range, 0.66% to 11% of SCDT, in patient samples. The long term variation of the method was evaluated using two control samples, with S CDT concentrations of 1.1 and 2%. Both controls showed low variation CV% 5.34 and 4.58, respectively (N=15), as analyzed over the whole trial period of three months. This compares favourably with the old method which showed a variation of CV% 6.99 and 5.46, for the low and high control, respectively. Figure 5: Correlation of the ‘old’ and ‘new’ method for the determination of CDT in serum. Conclusion The new method using the PLSAX 1000Å 8 μm 50 x 4.6mm ID column from Polymer Laboratories, showed improvements over the previous method. The new method has a reduced cycle time compared to the previously reported method thus leading to higher throughput and lower costs. Lower variability and thus improved reliability was also observed. References (1) B. Tandberg (2004) Laboratoriet, 2, 1011. (2) A. Helander et al. (2003) Clinical Chemistry, 48, 1881–1890. Presented at HPLC 07 0 min 12 tetra penta tri high di-sialo hexa THE HOUSE OF CHROMATOGRAPHY via C. Menotti, 11 20129 Milano Tel. 02.76.100.37 - 02.73.863.15 Fax 02.70.100.100 E-mail: [email protected] www.superchrom.it
Transcript of THE HOUSE OF CHROMATOGRAPHY via C. Menotti… · Quantitationof Carbohydrate DeficientTransferrinin...
Quantitation of Carbohydrate Deficient Transferrin in Serum: a New Method using Anion Exchange HPLCL. Lloyd3, R. Nilsson1, and A. Medin21 Dept of Clinical Chemistry, Växjö Hospital, Sweden2 Scantec Lab AB, Partille, Sweden3 Polymer Laboratories,
AbstractProlonged, excessive consumption of alcohol is known to affect the glycosylation states of proteins. In Sweden the presence of elevated levels of carbohydrate deficient transferrin, CDT, is an accepted clinical marker for disproportionate alcohol consumption. There are seven possible iso�forms of transferrin and the relative levels of these glycosylation states has been shown to be an indicator of excess alcohol consumption.
We present an improved anion exchange HPLC method capable of resolving the iso�forms of transferrin. Its potential suitability as an assay for the detection of alcohol abuse is demonstrated.
IntroductionHigh consumption of alcohol has several effects on the human body, which can be determined by laboratory analysis. Damage to cells can be estimated by the leakage of enzymes and is determined as serum levels of the liver enzymesglutamyl transferase (S�GT), alanine amino transferase (S�ALAT) and aspartateamino transferase (S�ASAT). The enzyme levels are known to increase as a result of high alcohol intake over time and although alcohol is not the only factor which can result in an increase in the serum levels of these enzymes, alcohol is the most common factor, and, so their measurement is used in the treatment of alcoholism and alcohol related disease. However, these tests do not give the specificity needed for the determination of alcohol abuse and so an alternative assay which has improved specificity is required.
A general effect on the glycosylation of proteins is observed with a high and prolonged exposure to alcohol and its metabolites. This effect is clearly seen withtransferrin, an iron transport protein, which has carbohydrate side chains that have terminal sialic acid residues. There are seven possible iso�forms of transferrin. In normal healthy individuals 80% of the protein is in its tetrasialo form, 14% in thepenta� and hexasialo forms, 5% in trisialo� and 1% is present as disialo transferrin.Asialo transferrin is not detected in normal healthy subjects but is found in 50% of alcoholics. An increase in the levels of asialo, mono� and disialo forms to over two percent has been correlated with a high daily intake of alcohol. The term carbohydrate deficient transferrin, CDT, relates to the total of these three forms. Carbohydrate deficient transferrin can be determined in plasma, P�CDT, urine, U�CDT, and serum, S�CDT and in Sweden it is the blood serum level of carbohydrate deficient transferrin, S�CDT, that is an accepted marker for extensive and prolonged consumption of alcohol1.
These sialic acids are charged and thus confer differences to the net protein charge depending on the number of sialic acids that are present as part of the protein. As a consequence of the different net charge the transferrin glycosylation variants can be successfully resolved using anion exchange chromatography (IEC). Recently, an HPLC method utilising anion exchange chromatography to quantify the disialoform was reported2. This method gives a specificity of 99% but does not quantify the individual form of S�CDT and has a relatively long cycle time.
It was anticipated by Roland Nilsson, Klin Kem Lab Växjö Lasarett, that for an assay further improvements could be obtained by quantification of all theglycosylation states and reducing the assay cycle time – improving the through�put. An improvement in the precision of the assay would be achieved if a gain in resolution could be obtained so that the individual forms could be quantified. The work presented here was carried out by Roland Nilsson and presents an assay for S�CDT which meets these criteria.
Växjö hospital where this work was carried out.
HPLC SystemColumn:PL�SAX 1000Å 5 μm, 50 x 4.6 mm IDEluent A: 20 mM BisTris, pH 6.4Eluent B: 20 mM BisTris, pH 6.2 + 0.25 M NaClEluent C: 2 M NaCl Gradient: 0 min 6% B
1 min 13% B4 min 24% B6.5 min 37% B6.6 min 38% B
Cleaning: 2 min with 100% CEqillibration: 4.4 min with 6% BFlow Rate: 1 mL/minTemperature: ambientDetection: UV, 460 nmInjection: 30 μLRun time: 9 min Cycle time: 13 min
Sample PreparationVenous 10 mL aliquots of blood were collected in vacuum tubes, centrifuged for ten minutes at 1200�1400 g, and the supernatant decanted into new tubes. From this 100 μL of serum was transferred into a 1.5 mL micro centrifuge tube and lipoproteins were precipitated by the addition of 20 μL “FeNTA” reagent* and 500 μL H2O. Then 20 μL of precipitating reagent** was added with gentle mixing and the tubes were left at 4 ˚C overnight. The tubes were centrifuged at 20000 g for 10 minutes after which 150 μL of the clear supernatant was transferred into 300 μL (12 x 32 mm) plastic vials.
*FeNTA reagent: 10 mM nitrilo acetic acid trisodium salt; FeCl3 in H2O
** Precipitating reagent: 50% 1 M MgCl2 solution in H2O / 50% 2% solution of Na�dextran sulphate in H2O (v/v).
ChromatographyA 30 μL aliquot of the prepared serum sample was used for each assay. A salt and pH gradient was used for the separation of the transferrin iso�forms, as detailed in the HPLC section. The run time was nine minutes and the total cycle time was 13 minutes. The glycosylation forms of the transferrin were separated based on the number ofsialic acid residues; the more sialic acid residues the longer the retention time (Figure 1) shows the chromatogram of a sample from a healthy individual.
Figure 1: Chromatogram showing the five sialo variants of transferrin in a ‘normal’sample, di�, tri�, tetra�, penta� and hexa�sialo.
Figure 2: Chromatogram showing the elution position of asialo transferrin.
Figure 3: Chromatogram showing the elution position of mono�sialo transferrin.
The anion exchange HPLC method is able to resolve the seven glycosylation forms of transferrin (Figures 1, 2 and 3). However, the a�sialo form is only seen in very rare cases and is of little diagnostic value as it occurs with elevated levels of the di�sialo form. The mono�sialo form also only occurs in very rare cases but is sufficiently well resolved to be identified during the assay.
0 min 12
tetra
penta
tri
di hexa
0 min 12
high di-sialo
a-sialo
0 min 12
mono-sialo
Data Calculation and InterpretationThe assay chromatograms are evaluated by assigning the five peaks correlating to theisoforms, di�, tri�, tetra�, penta�, and hex� sialo forms and calculating the areas of each peak. The areas for the individual peaks are then combined to give a measure of the totaltransferrins present. The S�CDT percent value is then calculated by dividing the area of the disialo form with the area for total transferrins. The normal result for S�CDT in healthy adults with low or moderate alcohol consumption is 1% S�CDT or lower (Figure 1). Values greater than 2% indicate high and continued intake of alcohol (Figure 4).
Figure 4: Chromatogram showing an enhanced level of di�sialo transferrin as would be expected from an adult sample after prolonged high intake of alcohol.
Results and DiscussionThis method was evaluated relative to the previously reported method using a regression study. Data from parallel analyses with both methods on routine patient investigations and analysis of control samples was also carried out over three months. A single PL�SAX 1000Å 8μm column was used throughout. The previous method used a 100 mm x 4.6 mm ID Source�15Q 4.6/100PE column packed with anion exchange material with a particle size of 15 μm.
The regression analysis in Figure 5 shows excellent correlation with the older method, slope = 1.01 and r = 0.9967, for the 78 patient samples analyzed. The results spanned the complete range, 0.66% to 11% of S�CDT, in patient samples.
The long term variation of the method was evaluated using two control samples, with S�CDT concentrations of 1.1 and 2%. Both controls showed low variation CV% 5.34 and 4.58, respectively (N=15), as analyzed over the whole trial period of three months. This compares favourably with the old method which showed a variation of CV% 6.99 and 5.46, for the low and high control, respectively.
Figure 5: Correlation of the ‘old’ and ‘new’ method for the determination of CDT in serum.
ConclusionThe new method using the PL�SAX 1000Å 8 μm 50 x 4.6mm ID column from Polymer Laboratories, showed improvements over the previous method. The new method has a reduced cycle time compared to the previously reported method thus leading to higher throughput and lower costs. Lower variability and thus improved reliability was also observed.
References(1) B. Tandberg (2004) Laboratoriet, 2, 10�11.(2) A. Helander et al. (2003) Clinical Chemistry, 48, 1881–1890.
Presented at HPLC 07
0 min 12
tetra
penta
trihigh di-sialo
hexa
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