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Articles |
1
Department of Clinical Chemistry, Ghent University Hospital, De Pintelaan 185, B9000 Ghent, Belgium.
2
Departments of Medicine and Chemical Pathology, Medical
School, The University of Zimbabwe, Avondale, Harare, Zimbabwe.
3
Department of Clinical Chemistry, Middelheim Hospital,
Lindendreef 1, B2020 Antwerp, Belgium.
4
Department of Clinical Chemistry, AZ Stuivenberg, Lange
Beeldekensstraat 267, B2060 Antwerp, Belgium.
5
Analis SA, 14 Rue Dewez, B5000 Namur, Belgium.
a Address correspondence to this author at: Department of Clinical Chemistry, De Pintelaan 185, B-9000 Gent, Belgium. Fax 32-9-240-4985; e-mail joris.delanghe{at}rug.ac.be.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: CZE was performed on a P/ACE 5000 using fused-silica capillaries [50 µm (i.d.) x 47 cm] and the CEOFIX CDT buffer system with addition of 50 µL of anti-C3c and 10 µL of anti-hemoglobin. Native sera were loaded by high-pressure injection for 3 s, separated at 28 kV over 12 min, and monitored at 214 nm.
Results: CDT was completely resolved by differences in migration times (di-trisialotransferrin, 9.86 ± 0.05 min; monosialotransferrin, 9.72 ± 0.05 min; asialotransferrin, 9.52 ± 0.04 min), with a CV of 0.15%. The number of theoretical plates was 312 000 ± 21 000 for the mono- and 199 000 ± 6500 for the di-trisialylated transferrin. Genetic CB and CD variants showed prominent peaks with migration times of 10.12 ± 0.06 and 9.89 ± 0.03 min, respectively, and the carbohydrate-deficient glycoprotein syndrome could be detected, excluding false-positive results. CZE results (as a percentage; y) correlated with the Axis %CDT TIATM (x) values by Deming regression analysis: y = 1.92x - 7.29; r = 0.89. CDT values in 130 healthy nonalcoholics were determined. The 2.5th and 97.5th percentiles were 1.84% and 6.79%.
Conclusions: CZE without sample pretreatment can determine CDT with good precision, allows detection of variants, and correlates with ion-exchange chromatography.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-glutamyltransferase (EC 2.3.2.2) and mean corpuscular volume have
low diagnostic sensitivity for detecting alcohol abuse before the stage
of organic complications and are indicators of disease in a particular
organ, with poor specificity for different etiological possibilities
(2)(3)(4).
Since the discovery that alcoholics demonstrate an abnormal transferrin
electrophoretic pattern (5), carbohydrate-deficient
transferrin
(CDT)1
has been considered the most valuable marker for monitoring
chronic alcohol abuse (2). Transferrin exhibits a
microheterogeneity in its iron, amino acid, and carbohydrate content.
Variations in iron content can be overcome by saturating samples with
iron (6), and amino acid variations are seen only in
rare genetic variants of transferrin (2). The commonly
occurring isoforms of diferric transferrin depend on variations in
carbohydrate content. Normal serum contains high concentrations of
tetrasialotransferrin (pI 5.4) and low amounts disialo-,
trisialo-, and pentasialotransferrin (pI 5.7, 5.6, and 5.2,
respectively) (2)(6)(7). In
alcoholics, transferrin fractions with di- (pI 5.7), mono- (pI 5.8),
and asialylated (pI 5.9) carbohydrate chains are increased
(8). It has been estimated that a minimum alcohol
consumption of 50–80 g/day for at least 1 week is needed to increase
the serum CDT in 
80% of subjects. The half-life of the marker is
16–17 days (2)(9)(10).
Early methods for CDT used isoelectric focusing (IEF) with laser densitometric quantification after immunoblotting (11). However, although this technique served as gold standard for the validation of most CDT procedures, IEF was too complex and laborious for routine clinical purposes. Alternative procedures were introduced based on anion-exchange chromatography (2)(12), chromatofocusing (13), HPLC (8) and fast protein liquid chromatography (14), but they also were complex and laborious. Recently, commercial methods for easy, time-saving CDT detection have been developed that use anion-exchange chromatography followed by RIA (CDTectTM and %CDT-RIATM), enzyme immunoassay (CDTect-EIATM), or turbidimetric immunoassay (%CDT-TIATM and ChronAlcoI.D.TM).
In 1989, capillary IEF was successfully applied to quantitative measurement of CDT (15). However, this method is not easily applied in a routine clinical environment. The problems in separation of transferrin isoforms using capillary zone electrophoresis (CZE) (16)(17)(18) could be resolved by an improved CZE method requiring only iron saturation as sample pretreatment (19)(20)(21). This opened prospects for application in a routine clinical laboratory.
In this study, we describe a new CZE method using the CEOFIX CDT buffer system (Analis, Namur, Belgium), which requires no sample pretreatment. Furthermore, influence of the presence of transferrin variants and carbohydrate-deficient glycoprotein syndrome (CDG) on the CDT analysis was evaluated. Finally, this method was compared with an ion-exchange chromatography-based method (%CDT-TIA; Axis Biochemicals).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sera from heterozygous individuals carrying the CD variant of transferrin were obtained from 2 healthy Caucasians and 11 Zimbabwean Bantus, an ethnic group with a high incidence of genetic CD transferrin variance. Sera from heterozygous individuals carrying the CB variant were obtained from two healthy Caucasians. Serum from a CDG patient was obtained from the Department of Pediatrics of the University Hospital, Gent.
cdt analysis
Capillary electrophoresis.
Analysis was carried out with a
P/ACE 5000 electropherograph (Beckman) with a single-wavelength
ultraviolet absorbance detector and an interference filter at 214 nm.
Uncoated fused-silica capillaries [50 µm (i.d.) x 47
cm] were purchased from Beckman. The buffer system of the CEOFIX CDT
method (Analis) was used. To 4.2 mL of running buffer, 50 µL
of anti-C3c antibodies and 10 µL of anti-hemoglobin (Dade Behring)
were added. The capillary was first rinsed for 1.2 min under pressure
with the "initiator" solution, which coated the capillary wall with
a polycation. This rinse was followed by a 1.5-min rinse with a
"buffer" solution containing a polyanion, which adds a second layer
of coating. Serum was loaded electrophoretically by pressure injection
for 3 s. Separation was performed over 12 min at a constant
voltage of 28 kV, and the peak areas were calculated by integration
software from Beckman. Between samples, the capillary was washed with
0.2 mol/L NaOH for 1.5 min, followed by water for 1 min.
Electrophoresis conditions were established for CDT analysis without
any sample pretreatment.
Turbidimetric immunoassay.
CDT was determined by the Axis
%CDT turbidimetric immunoassay (%CDT-TIA) according to the
manufacturer’s instructions. Briefly, serum transferrin is saturated
with Fe3+ before the low-sialic acid transferrin
(CDT) is separated by an ion-exchange chromatography minicolumn. The
eluate and the total transferrin are measured using the same
anti-transferrin antibodies on a Cobas Mira Analyzer (Roche
Diagnostics). The %CDT value is calculated, and values exceeding 6.0%
are considered increased.
quantification and cze performance characteristics
The peaks corresponding to transferrin isoforms were quantified as
the asialo-, monosialo-, di-trisialo-, tetrasialo-, and
pentasialotransferrin content as a percentage of the total transferrin
content, in terms of area under the curve. In common CC
transferrins, the CDT value was calculated by the addition of the peak
areas (%) of the asialo-, monosialo-, and
di-trisialotransferrins. In CD variants, the peak areas (%) of the
asialo-D, asialo-C, monosialo-D, monosialo-C, and di-trisialo-D
transferrins were used for CDT calculations. Capillary electrophoresis
separation characteristics were calculated according to the United
States Pharmacopoeia calculation method. Efficiency, expressed as
number of theoretical plates (N), was calculated as:
N =
16(t/W)2, where
t is the migration time, and W is the peak width
at the basis of the peak using tangent method. Resolution
(R) was calculated on the basis of the equation:
R = 2(t2 -
t1)/(W2
- W1), where
t2 and
t1 represent the migration times of
two adjacent peaks, and W2 and
W1 represent the widths at the
baseline of these two peaks. Peak asymmetry is calculated as the
tailing factor T =
W0.05/2f, where
W0.05 is the distance from the leading
edge to the tailing edge of the peak, measured at a point 5% of the
peak height from the baseline, and f is the distance from
the peak maximum to the leading edge of the peak at the position of 5%
peak height. The reproducibility of the peak area percentages and
migration times of the mono-, di-tri-, and tetrasialylated transferrin
peaks was tested by assaying the same pathological sample 22 times.
resolution of serum transferrin isoforms
Serum (50 µL) was incubated overnight with 100 µL of
anti-transferrin antiserum (Behring). After centrifugation at
2000g for 20 min, the supernatant was collected and
analyzed. The electropherographs of the serum before and after
immunosubtraction were compared to locate the transferrin peaks among
the bulk of serum proteins.
Resolution of the serum transferrin isoforms was performed by incubating 50 µL of serum with 0.01 U of neuraminidase (type V, from Clostridium difficile; EC 3.2.1.18; Sigma) at 37 °C. Serum from a healthy nonalcoholic subject was analyzed without incubation and after 0.5, 1, 3, 5, and 9 h of incubation. Sera from carriers of the CB and CD variants were analyzed without and after 0.5, 1, 3, and 5 h of incubation.
Interference from the C3c degradation products of complement C3 was investigated by comparing the results for a sample from a healthy subject analyzed after >2 h at room temperature using the CEOFIX CDT buffer (Analis) with and without the addition of anti-C3c antiserum (Behring) to the running buffer.
To investigate interference from sample instability, a fresh sample was analyzed within 2 h after venipuncture and then divided into three aliquots, which were stored refrigerated (4–8 °C), at room temperature (18–22 °C), and at 37 °C. Each aliquot was analyzed daily for 5 consecutive days, and the results were compared.
Iron saturation of transferrin was performed by incubating 1 mL of serum overnight with 25 µL of 500 mmol/L NaHCO3 and 18 µL of 10 mmol/L FeCl3. Serum samples from patients with iron deficiency anemia were analyzed with and without iron saturation before analysis. The capacity of on-column iron saturation through the presence of iron in the running buffer was evaluated by comparing the percentages of monosialo- and di-trisialotransferrin values for samples from patients with iron deficiency anemia analyzed without and with iron saturation before analysis.
statistics
Results are expressed as the mean ± SD. Within-run
imprecision was assessed by calculating the CV of the
peak area percentages and migration times of the monosialo-,
di-trisialo-, and tetrasialotransferrin peaks of the same pathological
sample. Peak area percentage CVs were calculated to asses the
between-day variation. The effect of on-column iron saturation was
evaluated by analyzing 10 samples from patients with iron deficiency
anemia with and without preanalysis iron saturation. Differences in the
monosialo- and di-trisialotransferrin peaks of samples saturated with
iron before analysis and native samples were evaluated using the paired
t-test. Deming regression analysis was used for the
comparison study between capillary electrophoresis and Axis %CDT-TIA.
The population of healthy nonalcoholic subjects was evaluated for
gaussian distribution by the Kolmogorov–Smirnov test. The
Student t-test and ANOVA were used to evaluate gender and
age differences in this population.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To demonstrate the ability of the proposed method to resolve all
of the CDT isoforms, serum was incubated with neuraminidase for
increasing times to obtain sequential cleavage of the sialic acid
moieties (Fig. 1
). In the electropherograph obtained without incubation,
tetrasialotransferrin (migration time, 10.05 ± 0.02 min) was the
dominant peak, with a moderate concentration of pentasialotransferrin
(migration time, 10.10 ± 0.06 min) and a low concentration of
di-trisialotransferrin (migration time, 9.86 ± 0.05 min). After
30 min of incubation, a decrease in the penta- and tetrasialo peaks and
an increase in the di-trisialo peak could be seen. With increasing
incubation times, monosialotransferrin (migration time, 9.72 ±
0.05 min) and asialotransferrin (migration time, 9.52 ± 0.04 min)
appeared and became the dominant peaks, whereas the penta-, tetra-, and
di-trisialic forms decreased and disappeared. After 9 h of
incubation, the asialylated peak became the dominant peak, and only a
trace of the monosialylated peak was present.
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Table 1
shows the CZE performance characteristics number of theoretical
plates, resolution, and peak asymmetry. Within-run reproducibility of
the absolute and relative (calculated as ratios of the migration time
of the tetrasialo peak) migration times and the peak area percentages
of the monosialo-, di-trisialo-, and tetrasialotransferrins from the
same pathological serum are included (Table 1
).
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Serum stored at least 2 h at room temperature after venipuncture was analyzed with and without anti-C3c in the running buffer. In the analysis without anti-C3c, the tail of a broad additional peak (migration time, 9.52 ± 0.02 min) interfered with the detection of asialotransferrin. Addition of anti-C3c antibodies to the running buffer eliminated the interfering peak, clearing the baseline of noise.
Storage of serum samples for 5 days did not interfere with the CDT measurement. The %CDT values were 3.59% ± 0.33% overall, 3.59% ± 0.44% after storage at 4–8 °C, 3.76% ± 0.42% after storage at room temperature, and 3.68% ± 0.32% after storage at 37 °C. The between-day CV was 12% for samples stored at 4–8 °C, 11% for samples stored at room temperature, and 8.8% for samples stored at 37 °C.
interference testing
Heterogeneity based on iron saturation.
The efficiency of
on-column iron saturation was evaluated by comparing the analysis of 10
samples from patients with iron deficiency anemia with and without iron
saturation before analysis. The differences in the percentages of
monosialo- and di-trisialotransferrin were 0.17% ± 0.32% (mean peak
area percentage, 1.70% ± 0.57%) and 0.06% ± 0.46% (mean peak area
percentage, 3.53% ± 1.29%), respectively. The paired
t-test revealed no significant difference in the percentages
of the monosialo- and di-trisialotransferrin peak areas. No peaks
migrating with the mobility of asialylated forms could be detected.
Identification of transferrin variants and CDG.
To determine
whether a healthy nonalcoholic carrier of a heterozygous CD
variant of transferrin would give false-positive CDT values in our
method, we compared the CZE pattern of CD variants with that of a
healthy nonabuser and an alcohol abuser (Fig. 2
). The transferrin CD variants showed a prominent tetrasialo-D
peak with a migration time (9.89 ± 0.03 min) approximating that
of the di-trisialylated C isoform. The heights of both the tetrasialo-C
and -D peaks were very similar. A small peak with a migration time
(9.75 ± 0.06 min) approximating that of the monosialo-C isoform
could be designated as di-trisialo-D transferrin. The pentasialo-D peak
migrated between the tetrasialo-D and tetrasialo-C peaks.
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To investigate the resolution of the isoforms in CD variants,
transferrin was gradually desialylated by increasing the incubation
time with neuraminidase. Analyses (Fig. 3A
) showed the subsequent appearance of the di-tri-, mono-, and
asialylated D and C isoforms. In most of the D variants, small
differences in migration times between the tetrasialo-D and the
di-trisialo-C isotypes, the di-trisialo-D and the monosialo-C isotypes,
and the monosialo-D and the asialo-C isotypes compromised individual
integration of each transferrin peak. CDT integration of CD variants,
including asialo-D, asialo-C, monosialo-D, monosialo-C, and
di-trisialo-D, remained possible; however, comigration of the
di-trisialo-C with the tetrasialo-D isoform excluded the di-trisialo-C
peak from CDT.
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To evaluate the influence of transferrin B isotypes on CDT analysis, we
compared the CZE pattern of healthy nonalcoholic CB variants with that
of a healthy nonabuser and an alcohol abuser (Fig. 2
). The analysis of
the CB variants demonstrated a prominent isoform with a migration time
(10.12 ± 0.06 min) approximating that of the pentasialylated C
isoform. The height of the tetrasialo-B peak was slightly higher than
that of the tetrasialo-C peak because of comigration of the
tetrasialo-B isotype and the penta-C-sialotransferrin. An additional
peak migrating slower (10.20 ± 0.05 min) than the pentasialo-C
isotype could be designated as the pentasialo-B transferrin (Fig. 2
).
Resolution of the isoforms in CB variants was investigated by analysis
after neuraminidase treatment. These experiments (Fig. 3B
) showed that
the desialylated B- and C-transferrin peaks have discrepant migration
times, doubling the number of transferrin peaks compared with the
analysis of the serum of a healthy nonalcoholic volunteer after
corresponding times of incubation with neuraminidase.
The electropherogram of the CDG patient revealed a minor
tetrasialylated peak and increased di-trisialylated (29.0%)
and monoglycosylated (5.4%) forms, leading to an increased CDT value
of 34.4% (Fig. 2
).
population distribution and method comparison
The analysis of 130 sera from healthy nonalcoholic subjects
demonstrated a gaussian distribution for the population according to
the Kolmogorov–Smirnov test (Fig. 4
). The 2.5th and 97.5th percentiles of the CDT values were
1.84% and 6.79% overall, 1.60% and 6.83% in the male population
(n = 69), and 2.32% and 6.70% in females (n = 61). No
significant gender or age difference was seen.
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Capillary electrophoresis %CDT (y) correlated with Axis
%CDT TIA (x) results in 77 serum samples of nonalcoholic,
healthy subjects (social drinkers were included) and patients admitted
to the Department of Psychiatry of the AZ Stuivenberg Hospital with
suspicion of chronic alcoholism: y =
1.92x - 7.29; r = 0.89; Fig. 5
. Deming regression revealed that in the low CDT range, CZE
results were generally lower than the Axis %CDT-TIA values, and in the
high range, CZE results were higher than the Axis %CDT-TIA values.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transferrrin has two iron-binding sites per molecule, and under
physiological circumstances serum transferrin is 30% saturated with
iron. Consequently, four different forms of transferrin with respect to
iron content can be distinguished in human serum: apotransferrin, and
N-terminal and C-terminal monoferric and diferric transferrin.
The differences in iron saturation produce differences in charge. The
electrophoretic migration of unsaturated transferrin overlaps the
variation in sialic acid content. Interference of apo- or monoferric
transferrin with CDT can be overcome by saturating the serum with iron
(29)(30). Until now, the saturation has been
performed before separation (precolumn mode) because it allows
flexibility in optimizing reaction conditions and no complex
instrumentation is needed. In the present CDT method, the iron is
complexed to transferrin during the electrophoretic separation
(on-column mode); the capillary column serves as the reactor vessel.
Once injected onto the column, the sample is allowed to react with the
iron present in the running buffer. In the on-column method, sample
dilution is reduced compared with the precolumn complexation mode,
making the on-column approach especially suitable for the analysis of
biological samples that are already very dilute. Furthermore,
introduction of a low-ionic strength sample into the iron-containing
running buffer, which has a high ionic strength, induces sample
stacking, increasing the resolution. In precolumn saturation, reducing
the ionic strength of the sample by dilution can produce stacking;
however, it requires long injection times for sufficient resolution
(21). To obtain optimal complexation, the saturation should
be fast and complete, no interfering side products should be formed,
and the iron saturation should be stable. In vitro studies of iron
release from transferrin revealed that acidification together with the
reduction of Fe(II) ions to the Fe(III) state, at the cost of
intracellular conversion of NADH to NAD+, are
necessary in the release of iron from transferrin (30)(31)(32).
The alkaline pH of the running buffer prevents the dissociation of iron
from transferrin during electrophoresis. Our experiments on
iron-deficient serum samples demonstrate sufficient transferrin
iron-load stability.
Genetic variants of human serum transferrin were first discovered in
1957 by Smithies (33). In addition to the common (C)
transferrin type, a series of anodal (B) and cathodal (D) variants have
been reported in different human populations
(7)(11)(34). The degree of genetic
polymorphic variation in transferrin is limited in Caucasians. Most
individuals express the C allele, and rarely, heterozygous CB variants
can be seen. In African Americans, the transferrin CD phenotype has a
frequency of 10% (35), and the highest frequencies of
the D allele are seen in West Africa and western stream Bantu
(36).
Our experiments demonstrated that CB and CD variants are easily detected by CZE analysis based on differences in migration times and the high similarity in peak heights between the C and B or D peaks. Neuraminidase treatment demonstrated that in CB variants, the respective carboxylated isoforms of the C and B isotypes migrated with different times so that peak integration of each isotype remained possible, but with loss of resolution. In CD variants, integration of the individual isotypes was compromised, and CDT was calculated as the sum of the di-trisialo-D, monosialo-C, monosialo-D, asialo-C, and asialo-D transferrins to avoid the risk of false-positive CDT values. Because the di-trisialo-C isoform is included in the non-CDT transferrin, reference values for D variants have to be established. IEF and HPLC allow detection of genetic variants, but methods using anion-exchange chromatography might give falsely increased CDT values in CD variants and miss the detection of variants because the eluted CDT fraction is quantified without visualization of the transferrin pattern (2)(4)(26). The coelution of tetrasialo-D with trisialo-C isoforms might cause strong overdetermination of CDT. Therefore, mainly anion-exchange chromatography-based tests that include trisialotransferrins are at risk for false-positive results (28). Recently, a comparison between CZE and immunoturbidimetry showed that the serum transferrin concentration in D variants might be underestimated when analyzed immunologically (37). Although transferrin concentrations in D variants were comparable to CC transferrin concentrations when measured by immunoturbidimetry, CZE areas under the curve differed significantly. We included the trisialylated isoform in addition to the asialylated, monosialylated, and disialylated isoforms in our CDT value because the metabolic process that converts high- to low-sialic acid transferrin is considered a continuous process and trisialotransferrin is increased in alcoholics (36). Furthermore, measurement of the different transferrin fractions separately could give additional information because disproportionate alcohol-induced increases in the different glycosylated isoforms might occur and additional information regarding the alcohol consumption over time could be provided. The clinical performance of separate fractions requires further evaluation.
In CDG patients, one-half of the transferrins lack two or all four of their terminal carbohydrate groups (38)(39). Like IEF and HPLC (2)(4)(26), CZE allows detection CDG. Our findings of markedly increased CDT in the serum of a CDG patient confirm previous observations (17). Recently, increased CDT values and abnormal protein glycosylation were demonstrated in a patient with achondroplasia, demonstrating the challenge in CDT determinations (40).
The comparison of CDT measurements by CZE and an ion-exchange chromatographic method (Axis %CDT-TIA) revealed a significant correlation. Although the Axis %CDT-TIA method includes only 50% and our CZE method 100% of the trisialotransferrin, lower CZE values compared with the Axis results were seen in the lower CDT range. Comparable results have been described for IEF (41) and CDTect (42). In spite of the high sensitivity of the Axis %CDT-TIA (41) method and the inclusion of only one-half of the trisialotransferrin, no %CDT values <3% could be obtained in our study or in previously reported data (41)(42)(43). The high correlation between the Axis %CDT-TIA and %CDT HPLC (41) was obtained because the %CDT-TIA minicolumn performance was calibrated so that the correlation between HPLC and TIA methods was at a maximum (44). This calibration produced an artificial increase in the Axis %CDT-TIA values in the lower region.
In conclusion, CZE is sufficiently sensitive to determine CDT isoforms with good precision and high resolution. The present method provides a CDT determination without any sample pretreatment, reducing manipulation errors and eliminating unnecessary dilution steps. Furthermore, we showed that CZE can easily detect genetic variants and CDG, reducing false-positive values, and that it is correlated with an ion-exchange chromatography-based method.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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