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Articles |
1
Zentrum für Innere Medizin, Abteilung Kardiologie, Baldingerstrasse, Philipps-Universität, D-35033 Marburg, Germany.
2
bioscientia, Institut für
Laboruntersuchungen Ingelheim GmbH, Konrad-Adenauer-Strasse 17, D-55218
Ingelheim, Germany.
3
Abteilung Klinische
Chemie und Pathobiochemie-Zentrallaboratorium, Baldingerstrasse,
Philipps-Universität, D-35033 Marburg, Germany.
4
Abteilung Innere Medizin und Zentrallabor, St.
Nikolaus-Stiftshospital, D-56626 Andernach, Germany.
a Author for correspondence. Fax 049-6132-781-428; e-mail arndt{at}bioscientia.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: Isoelectric focusing on the PhastSystemTM followed by immunofixation, silver staining, and densitometry was used to validate the initial transferrin isoform fractionation step on anion-exchange microcolumns involved in the ChronAlcoI.D.TM assay.
Results: The in vitro transferrin iron load was complete and
stable. The CDT and non-CDT transferrin fractionation on anion-exchange
microcolumns was reliable and reproducible (CV 
10%). Except for
quantitatively unimportant traces of
trisialo-Fe2-transferrin (<5% of total CDT), only
asialo-, mono-, and disialo-Fe2-transferrin were detected
in the microcolumn eluates (n = 170). There was a loss of
proportionally similar amounts of asialo-Fe2-transferrin
(during column rinsing) and disialo-Fe2-transferrin (on the
anion exchanger). Thus, the peak height ratios for disialo- and
asialo-Fe2-transferrin did not change from >1 (serum) to
<1 (eluates) as described for the CDTect assays. The transferrin
patterns in the ChronAlcoI.D. eluates were representative of
those in serum. Transferrin D variants with isoelectric points close to
that of trisialo-Fe2-transferrin C1 did not cause
overdetermination of CDT by the ChronAlcoI.D. test.
Conclusions: The initial CDT and non-CDT fractionation step involved in determination of CDT by the ChronAlcoI.D. assay is efficient for eliminating non-CDT transferrins from serum before quantification of CDT in the final turbidimetric immunoassay. We recommend IEF for validation of other (commercial) CDT analysis methods and of odd CDT results.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The aim of our study was to assess which transferrin isoforms are measured as CDT by the ChronAlcoI.D. assay. The following points, which are crucial for the specificity of the initial CDT and non-CDT transferrin isoform fractionation step, were investigated: (a) the efficiency of the in vitro transferrin iron-saturation step (which is used to establish a unique transferrin iron load by formation of Fe2-transferrins and elimination of Fe1- and Fe0-transferrins); (b) the transferrin iron load stability during passage over the anion-exchange columns to prevent coelution of non-CDT and CDT transferrins with differing iron and sialic acid content but identical pI values; (c) the efficiency of the initial CDT and non-CDT transferrin fractionation step on the anion-exchange microcolumns; (d) the reproducibility of the anion-exchange microcolumn separation; and (e) possible effects of genetic transferrin D variants on the ChronAlcoI.D. assay results. Knowing which transferrin isoforms, and to what extent they are measured as CDT by the CDT analysis tests currently available, will undoubtedly be helpful for further studies on the pathomechanisms of CDT increases and for the diagnostic efficiency of CDT as the most specific marker of chronic alcohol abuse at present.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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serum samples
All procedures were performed in accordance with the Helsinki
Declaration of 1975, as revised in 1996.
Only surplus serum sample volumes from routine investigations were used. Blood was drawn after overnight fasting into sterile gel tubes (Sarstedt). The gel barrier consisted of a polymerized acrylic resin that does not affect the serum CDT concentration (18). After clotting at room temperature for 30 min, the blood samples were centrifuged at 2000g for 10 min at 4 °C. Serum was removed immediately with disposable pipettes (to avoid contamination with microorganisms); aliquots were transferred into sterile, leak-proof plastic containers (Micro Tubes with screw cap; Sarstedt) and stored at -22 °C until the day of analysis.
ChronAlcoI.D. ASSAY
Serum CDT concentrations and the CDT/transferrin ratios were
determined by the ChronAlcoI.D. assay in accordance with the
instructions of the manufacturer. The test comprises the following
steps:
In vitro transferrin iron saturation.
Serum or control sample
(100 µL) and ferric saturation reagent (500 µL) are mixed and
incubated for 5–10 min at room temperature.
Anion-exchange microcolumn separation of CDT- and
non-CDT-transferrins.
The Fe3+-saturated
serum or control sample (500 µL) is pipetted directly onto the top of
the filter of the microcolumn. The sample drains until the top filter
appears dry, and the effluent (load step) is discarded (non-CDT
transferrins and CDT transferrins adsorb to the anion exchanger). The
column is rinsed with 1.5 mL of elution buffer, and the effluent (rinse
step) is discarded. An additional volume of 2.5 mL of elution buffer is
pipetted onto the column filter, the buffer drains until the filter
appears dry, and the eluate is collected in appropriately labeled test
tubes (CDT transferrins are eluted).
Preparation of the total transferrin solution.
The total
transferrin solution is prepared while microcolumn separation is taking
place. The Fe3+-saturated serum or control sample
(20 µL) is mixed with 800 µL of elution buffer. This sample is used
in the following turbidimetric immunoassay for determination of the
total serum transferrin concentration.
Quantification of CDT and total transferrin by a microtiter-plate
turbidimetric immunoassay.
Calibrators, microcolumn eluates, and
total transferrin dilution samples (200 µL of each) are pipetted
directly into the bottom of each well, and atypical absorbance
(background) is read at 405 nm (Dynatec MR 5000 reader; Dynex
Technologies). Transferrin antibody solution (100 µL) is then added
to each well. After gentle agitation for 15 min at room temperature,
the absorbance is read at 405 nm. The analysis data were evaluated with
Dynex Revelation 3.2 software (Dynex Technologies). The results were
reported as CDT/transferrin ratios as well as CDT concentrations.
Quality control for the whole ChronAlcoI.D. assay was in accordance with the guidelines of the German Federal Medical Association. Within each assay, serum pool aliquots with CDT values near the cutoff of 2.5–2.7% for women and men (17), and two control samples with normal and increased CDT ratios (CDT control set; DPC/Biermann) were used for internal quality control. The control samples were placed at the beginning and the end of each set of samples. Control and serum samples were analyzed in duplicate. The laboratory regularly participates in external quality-control programs.
ief, immunofixation, and silver staining
The efficiency of the in vitro iron-saturation step, the
transferrin iron load stability during passage through the
microcolumns, the efficiency and reproducibility of the fractionation
of CDT and non-CDT-transferrins, and the possible effects of
transferrin D variants were assessed by IEF analysis of the
transferrin isoform patterns in the corresponding sample aliquots after
each intermediate step of the ChronAlcoI.D. CDT and non-CDT
fractionation procedure. We investigated (a) the serum
samples after Fe3+-transferrin saturation (Fig. 1a
, lanes A, and Fig. 3
); (b) the column effluents after
application of the iron-treated serum samples to the top of the
anion-exchange microcolumns; (c) the column effluents after
the columns were rinsed with 1.5 mL of elution buffer (Fig. 1a
, lanes
B); (d) the column eluates after the addition of 2.5 mL of
elution buffer to the column (this eluate usually is used for
quantifying CDT in the final turbidimetric immunoassay; Fig. 1a
, lanes
C; Fig. 2
, lane 13; Figs. 3
and 4
); and (e) additional 2
mol/L NaCl eluates that were used for recovery studies (usually not
part of the original ChronAlcoI.D. test; Fig. 1a
, lanes D).
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Because of the limited capacity of the IEF system, it was impossible to analyze all samples immediately after the original ChronAlcoI.D. step. Thus, serum samples and column effluents and eluates were frozen immediately and stored at -22 °C until analysis (usually within 2 weeks). This sample storage (which is not part of the ChronAlcoI.D. assay, but which was part of our experimental setup) caused a partial loss of transferrin iron in the effluents and eluates, probably because of the ionic strength of the buffers used. To these samples, additional amounts of Fe3+ were added, according to Hackler et al. (8). Identical IEF transferrin patterns were obtained when we analyzed fresh samples and the same samples after freezing and additional Fe3+ treatment in parallel. This demonstrates that our procedure is appropriate for readjusting the original complete transferrin iron load in thawed column effluents and eluates.
IEF.
IEF was performed on the
PhastSystemTM, followed by immunofixation and
silver staining as described by Hackler et al. (8) with the
modifications described by Arndt et al. (19). In short,
polyacrylamide gels, pH 5–6 (43 x 50 x 0.45 mm; total
acrylamide content, 5%; cross-linker content, 3%; Pharmalyte
5–6® diluted 1:16, by volume), adhered to a
plastic support film (GelBondTM PAG film;
Biozym-Diagnostik), were prepared in house and prefocused for 75 V-h.
Using the Sample ApplicatorTM 8/1
(Pharmacia/LKB), we applied eight samples (1 µL of each sample). The
sample applicator was inserted into the most cathodic position of the
sample applicator arm. Sample application was performed for 15 V-h, and
the separation was performed for 200 V-h.
Immunofixation.
Immunofixation was done immediately after the
IEF (20). The gels were covered with 175 µL of polyclonal
IgG antibodies to transferrin (50 µL of antibody diluted in 150 µL
of 150 mmol/L NaCl) and incubated at room temperature in a
moist chamber for 40 min. Unprecipitated (non-transferrin) proteins
were removed by washing the gels with 150 mmol/L NaCl overnight with
vigorous agitation.
Silver staining.
Silver staining was carried out in the
PhastSystem Development UnitTM according to
Hackler and Kleine (20) with the following modification: The
staining reaction was stopped by incubating the gel in 50 mmol/L EDTA
(instead of 50 mL/L acetic acid). The gels were washed in
deionized water for 2 h, dried in air, and kept for documentation.
Transferrin bands were identified by parallel analysis of a
cerebrospinal fluid (CSF) sample in each gel, showing (physiologically)
asialo- to hexasialo-Fe2-transferrin isoforms.
Densitometry.
Densitometry was performed on a Preference
densitometer (Sebia).
In adjusting the IEF sensitivity for detection of the CDT-transferrins,
an overload of the tetrasialo-Fe2-transferrin
fraction was accepted. Thus, the intensity (Fig. 1a
) and peak height or
peak area (Fig. 1b
) of this
tetrasialo-Fe2-transferrin fraction did not
correlate with the transferrin content. In contrast to
tetrasialo-Fe2-transferrin, asialo-, mono-, di-,
and trisialo-Fe2-transferrin fractions were not
overloaded. Thus, the peak heights or areas of these fractions could be
used for determination of the percentage of the
trisialo-Fe2-transferrin contamination from total
CDT.
other assays
To achieve comparable transferrin band and peak intensities
between lanes A (serum) and D (2 mol/L NaCl eluate) of Fig. 1
, the
samples were diluted to a uniform total transferrin concentration of 8
mg/L. The total transferrin was determined with a Turbitimer and
Turbiquant® transferrin reagent (Dade Behring).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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a, lanes C) were investigated together with the corresponding
Fe3+-treated serum samples, and 36 were
investigated together with the corresponding serum samples (Fig. 1a
, lanes A), column effluents (rinse step; Fig. 1a
, lanes B), and 2 mol/L
NaCl eluates (Fig. 1a
, lanes D); for the nomenclature, see
Materials and Methods. As the main result of our study, we
found only CDT transferrin isoforms (asialo-, monosialo-, and
disialo-Fe2-transferrin) in the ChronAlcoI.D.
microcolumn eluates, except for quantitatively unimportant traces
(<5% of total CDT) of trisialo-Fe2-transferrin.
In testing the different points discussed earlier, we obtained the
following results.
in vitro transferrin iron saturation
When in vitro transferrin iron saturation is complete, only
Fe2-transferrin bands, but not
Fe0- and Fe1-transferrin
bands, should appear in the transferrin IEF band pattern. Furthermore,
the transferrin band pattern should be unaffected by an additional
(second) Fe3+-transferrin saturation step. We
tested 98 serum samples. Typical transferrin isoform band patterns
obtained by IEF after Fe3+-transferrin saturation
are shown in lanes A of Fig. 1a
and lane " Serum" of Fig. 3
.
Fe2-transferrin bands, but not
Fe0- and Fe1-transferrin
bands, were detected. The bands corresponded to di-, tri-, tetra-,
penta-, and hexasialo-Fe2-transferrin (from
cathode to anode) as verified by comparison with lane "CSF" in
Figs. 1a
and 3
. The more intense bands of
disialo-Fe2-transferrin and additional bands of
monosialo- and asialo-Fe2-transferrins seen in
lanes A of Fig. 1a
from the alcoholics with homozygous transferrin C1
[alcoholic (Tf C1)] and heterozygous transferrin C1D [alcoholic (Tf
C1D)] phenotypes in comparison with lane A of the control [control
(Tf C1)] are attributable to the chronic alcohol abuse of these
patients. The complex transferrin isoform band patterns in Fig. 1a
, lanes A–D of the alcoholic with transferrin C1D [alcoholic (Tf C1D)]
are explained further below.
The additional amounts of Fe3+ added to the samples according to the method of Hackler et al. (8) did not affect either the number of transferrin bands or the positions of the transferrin bands within the gel. From this it follows that the ChronAlcoI.D. Fe3+-transferrin saturation step is effective in achieving a uniform transferrin iron load by complete elimination of Fe0- and Fe1-transferrins.
transferrin iron load stability during passage over the
anion-exchange microcolumn
Transferrin iron loss, and thus reformation of
Fe0- and Fe1-transferrins,
can occur under nonoptimal pH conditions during the analytical process
(8). This loss can cause distinct overdetermination of CDT
because of coelution of transferrin isoforms with differing sialic acid
and iron content but the same pI (8). Thus, we checked the
2.5-mL eluates (which are used in the final CDT quantification step of
the ChronAlcoI.D. assay) for the presence of Fe0-
and Fe1-transferrins immediately after the
elution step and after additional amounts of Fe3+
were added according to the method of Hackler et al. (8).
Altogether, 170 column eluates (which are used in the final
turbidimetric immunoassay involved in the ChronAlcoI.D. test) were
assessed (lanes C of Fig. 1a
; lane 13 of Fig. 2
; lanes "Eluate" of
Fig. 3
; and lanes 1–7 of Fig. 4
). When fresh effluents and eluates (as
obtained by the original ChronAlcoI.D. procedure) were analyzed,
Fe0- and Fe1-transferrins
were not detected. The added Fe3+ did not affect
the transferrin band patterns, which demonstrates a complete and stable
transferrin iron load during passage over the microcolumn. Examination
of the column effluents and eluates only a few hours after the elution
step showed a small transferrin iron loss (weak bands of
Fe0- and Fe1-transferrins).
The loss was marked after storage of the eluates for 3 months
at -22 °C. After we added Fe3+ to these
samples (to readjust the original transferrin iron load), we could no
longer detect bands of Fe0- and
Fe1-transferrin or of more highly sialylated
non-CDT transferrins by IEF. This demonstrates that the transferrin
iron loss occurred during the sample storage, but not during the CDT
and non-CDT fractionation on the anion-exchange microcolumns. To test
this point further, we determined the CDT/transferrin ratio and the CDT
concentration from column eluates of seven serum samples with normal
and increased CDT after storage for 1, 2, 3, and 6 days at 4–8 °C
without adding further amounts of Fe3+.
The corresponding CVs were between 3.2% and 10%, which is
close to the imprecision of the ChronAlcoI.D. assay reported by Arndt
et al. (17). We conclude that the transferrin iron
saturation in accordance with the test instructions of the
ChronAlcoI.D. assay was complete and stable during the CDT and non-CDT
transferrin fractionation procedure. It permits reliable elimination of
Fe0- and Fe1-transferrins
and thus a uniform Fe2-transferrin iron load in
serum samples. The column eluates can be stored at 4–8 °C overnight
without the risk of false CDT results in the final ChronAlcoI.D.
turbidimetric immunoassay.
cdt and non-cdt transferrin fractionation
This part of the assay consists of three intermediate steps:
application of the Fe3+-treated serum to the
microcolumn, rinsing the column, and eluting the CDT transferrins. The
specificity of the microcolumn separation was tested by IEF of the
transferrin isoforms occurring in these matrices. We analyzed 6 column
effluents from the loading step, 36 column effluents from the washing
step, and 170 column eluates. We did not detect transferrins in the
column effluent from the loading step, confirming that there was
complete adsorption of all transferrin isoforms to the anion exchanger
(not shown). Distinct amounts of
asialo-Fe2-transferrin appeared in all column
effluents during the wash step, regardless of whether normal or
increased amounts of CDT appeared in the serum sample (Fig. 1a
, lanes
B; Fig. 2
, lanes 5 and 6), revealing that this CDT transferrin was
partially lost when the microcolumns were rinsed. The column eluates
(which are used for the final CDT quantification) contained asialo-,
mono-, and disialo-Fe2-transferrin, which
collectively are referred to as CDT (16), and traces of
trisialo-Fe2-transferrin (Fig. 1a
, lanes C; Fig. 2
, lane 13; Fig. 3
, lane "Eluate"; Fig. 4
). The recovery of CDT was
tested by eluting the microcolumns with 1 mL of 2 mol/L NaCl (the
optimal volume for complete elution of all transferrin isoforms from
the anion exchanger was assessed by fractionated elution in 500-µL
steps). The 2 mol/L NaCl eluates contained mainly more highly
sialylated, non-CDT transferrins (tri-, tetra-, penta-, and
hexasialo-Fe2-transferrin) but also
disialo-Fe2-transferrin. The latter finding
reflects a partial retention of this CDT transferrin by the anion
exchanger. Altogether, we found a partial loss of
asialo-Fe2-transferrin in the column rinse step
and a partial loss of disialo-Fe2-transferrin on
the anion exchanger. This incomplete recovery of CDT was observed for
serum samples with normal as well as with increased CDT concentration.
We tested by fractionated rinsing and fractionated elution whether the
elution buffer volumes for rinsing the microcolumns and eluting the CDT
transferrins, and thus the CDT recovery, could be optimized (Fig. 2
). As shown in Fig. 2
, an elution buffer volume of 1.0 mL
(instead of 1.5 mL) would be sufficient for rinsing the column. With
this volume, asialo-Fe2-transferrin would be
completely retained on the anion exchanger (Fig. 2
, lanes 1–4). Rinse
volumes >1.0 mL, e.g., 1.5 mL in accordance with the test
instructions, cause a partial loss of
asialo-Fe2-transferrin (Fig. 2
, lanes 5 and 6).
Increasing the buffer volume for eluting the CDT transferrins from 2.5
mL (original volume) to 3.0 mL (Fig. 2
, lane 12) would improve the
recovery of disialo-Fe2-transferrin, but at the
same time it would exacerbate the coelution of
trisialo-Fe2-transferrin. Reducing the elution
buffer volume from 2.5 mL to 2.0 mL allows almost complete retention of
trisialo-Fe2-transferrin (Fig. 2
, lane 10), but
it also allows higher loss of
disialo-Fe2-transferrin on the anion exchanger.
To assess the extent of CDT loss by the ChronAlcoI.D. transferrin
isoform fractionation step, we diluted serum samples and the
corresponding microcolumn eluates (which are used in the final
turbidimetric immunoassay) to obtain similar peak heights for
disialo-Fe2-transferrin (Fig. 3
). Taking into account the different dilution factors, the CDT
loss was estimated to be 
30%. We also used these samples to test
whether the CDT transferrin patterns in the eluate are representative
of those in the iron-treated serum samples. Comparing the serum
disialo-Fe2-transferrin/asialo-Fe2-transferrin
peak height ratios with those of the corresponding ChronAlcoI.D. eluate
(Fig. 3
) showed that the transferrin isoform patterns in the
ChronAlcoI.D. column eluates are representative of the transferrin
isoform pattern in the serum sample. The almost identical
disialo-Fe2-transferrin/asialo-Fe2-transferrin
peak height ratios in serum (3.2) and eluate (3.4) indicate a
proportionally similar loss of
asialo-Fe2-transferrin (during column rinsing)
and disialo-Fe2-transferrin (on the anion
exchanger). Thus, the
disialo-Fe2-transferrin/asialo-Fe2-transferrin
peak height ratios did not change from >1 (serum) to <1 (eluate) as
described for the CDTect assays (13). Identical results were
obtained for serum samples with normal and increased CDT concentration
(Figs. 1
and 3
). An important fact is that increased CDT fractions in
serum were always reflected in the corresponding microcolumn eluates.
This is another indication that the ChronAlcoI.D. microcolumn eluates
are representative of the serum sample. Altogether, the original
ChronAlcoI.D. CDT and non-CDT transferrin fractionation procedure
yields reliable separation and thus specific determination of CDT in
the final turbidimetric immunoassay. The elution buffer volume of 2.5
mL seems to be a good compromise between maximum analytical specificity
(exclusion of more highly sialylated non-CDT transferrins, e.g.,
trisialotransferrin and, most important, tetrasialotransferrin) on the
one hand and maximum recovery of CDT on the other hand.
reproducibility of the microcolumn performance
The within-run reproducibility of the whole CDT and non-CDT
transferrin fractionation step was tested by processing seven aliquots
of the same serum sample with increased CDT using seven different
columns in one analytical run. When we calculated the means,
SDs, and CVs of the peak height ratios of
disialo-Fe2-transferrin/
monosialo-Fe2-transferrin,
disialo-Fe2-transferrin/asialo-Fe2-transferrin,
and
monosialo-Fe2-transferrin/asialo-Fe2-transferrin
for each serum sample, we obtained imprecision values (CVs) of 5%,
9%, and 10%, respectively (Fig. 4
), which were comparable to the intraassay imprecision (9%) of
the whole ChronAlcoI.D. (17).
effects of transferrin d variants on the determination of cdt by
the ChronAlcoI.D. ASSAY
Transferrin D variants have been reported to interfere with the
determination of CDT, producing false positives with respect to chronic
alcohol abuse (7). We analyzed serum samples of an alcoholic
and a healthy proband with heterozygous transferrin D phenotypes. The
genetic transferrin D variants had isoelectric points very close to
that of trisialo-Fe2-transferrin C1. The
transferrin isoform band patterns in serum, effluent (rinse step),
eluate, and NaCl eluate of the alcoholic are shown in Fig. 1a
[alcoholic (Tf C1D)]. Because both transferrin C1 and transferrin D
also appear as transferrin isoforms with different sialic acid
contents, the IEF transferrin isoform band patterns were complex. The
transferrin bands belonging to transferrin C1 are indicated by dots to
the left and those of transferrin D variant are indicated by dots to
the right of the bands [Fig. 1a
, alcoholic (Tf C1D)]. The open
circles in lanes A and D of the alcoholic (Tf C1D) pattern
indicate the tetrasialo-Fe2-transferrin
fractions of transferrin C1 (open circle to the left of the band) and
transferrin D (open circle to the right of the band). The transferrin
isoform band pattern in serum after in vitro transferrin
Fe3+ saturation is shown in lane A. Distinct
bands of disialo-Fe2-transferrin and additional
bands of mono- and asialo-Fe2-transferrin in
comparison with lane A of the control (Fig. 1a
) reflect the chronic
alcohol abuse of this patient.
The IEF transferrin band pattern in the column effluent (rinse step) is
shown in lane B of the alcoholic (Tf C1D) pattern in Fig. 1a
.
The two bands represent asialo-Fe2-transferrin D
(most cathodic and more intense band) and
asialo-Fe2-transferrin C1 (more anodic band).
Lane B shows that larger amounts of the
asialo-Fe2-transferrin D in comparison with
asialo-Fe2-transferrin C1 were lost when the
column was rinsed. Lane C of the alcoholic (Tf C1D) pattern in Fig. 1a
shows the transferrin isoform pattern in the column eluate (CDT elution
step). The band pattern is complex, with two distinct bands
corresponding to disialo-Fe2-transferrin C1 (more
anodic band) and disialo-Fe2-transferrin D (more
cathodic band). The first overlaps with traces of
trisialo-Fe2-transferrin D, the latter with
monosialo-Fe2-transferrin C1. There are also
traces of trisialo-Fe2-transferrin C1 (most
anodic band), monosialo-Fe2-transferrin D,
asialo-Fe2-transferrin C1, and
asialo-Fe2-transferrin D (from anode to cathode).
For determination of CDT from this sample, it is important that
tetrasialo-Fe2-transferrin of the transferrin D
variant does not appear in the column eluate (Fig. 1a
, alcoholic (Tf
C1D), lane C). Coelution of
trisialo-Fe2-transferrin D in the column eluate
might cause an overdetermination of CDT. However, the loss of larger
amounts of asialo-Fe2-transferrin D during column
rinsing [Fig. 1a
, lane B of the alcoholic (Tf C1D) pattern]
compensates partially for this lack of specificity. Indeed, we
determined a CDT/transferrin ratio of 2.3% and a CDT concentration of
83 mg/L for the healthy proband with the transferrin C1D phenotype.
Both values were below the corresponding cutoffs of 2.5–2.7% and
100–110 mg/L (17). For the alcoholic, we measured a
CDT/transferrin ratio of 12% and a CDT concentration of 399 mg/L.
Altogether, the individual alcohol consumption of these two persons was
correctly reflected by the ChronAlcoI.D. results (despite the presence
of transferrin D variants).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In conclusion, with correct use, the initial CDT and non-CDT fractionation step involved in determination of CDT by the ChronAlcoI.D. assay is an efficient procedure for the elimination of non-CDT transferrins from the serum sample before CDT quantification in the final turbidimetric immunoassay. A standardization of CDT analysis is urgently needed.
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Acknowledgments |
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Footnotes |
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References |
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The following articles in journals at HighWire Press have cited this article:
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  T. Arndt Asialotransferrin--An Alternative to Carbohydrate-deficient Transferrin? Clin. Chem., June 1, 2003; 49(6): 1022 - 1023. [Full Text] [PDF]
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 M. J. Schwarz, I. Domke, A. Helander, P. M. W. Janssens, J. van Pelt, B. Springer, M. Ackenheil, K. Bernhardt, G. Weigl, and M. Soyka MULTICENTRE EVALUATION OF A NEW ASSAY FOR DETERMINATION OF CARBOHYDRATE-DEFICIENT TRANSFERRIN Alcohol Alcohol., May 1, 2003; 38(3): 270 - 275. [Abstract] [Full Text] [PDF]
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F. J. Legros, V. Nuyens, M. Baudoux, K. Zouaoui Boudjeltia, J.-L. Ruelle, J. Colicis, F. Cantraine, and J.-P. Henry Use of Capillary Zone Electrophoresis for Differentiating Excessive from Moderate Alcohol Consumption Clin. Chem., March 1, 2003; 49(3): 440 - 449. [Abstract] [Full Text] [PDF]
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F. de Loos, K. M.L.C. Huijben, N. C.A.J. van der Kar, L. A.H. Monnens, L. P.W.J. van den Heuvel, J. E.M. Groener, R. A. de Moor, and R. A. Wevers Hemolytic Uremic Syndrome Attributable to Streptococcus pneumoniae Infection: A Novel Cause for Secondary Protein N-Glycan Abnormalities Clin. Chem., May 1, 2002; 48(5): 781 - 784. [Full Text] [PDF]
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A. Helander, G. Eriksson, H. Stibler, and J.-O. Jeppsson Interference of Transferrin Isoform Types with Carbohydrate-deficient Transferrin Quantification in the Identification of Alcohol Abuse Clin. Chem., July 1, 2001; 47(7): 1225 - 1233. [Abstract] [Full Text] [PDF]
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T. Arndt Carbohydrate-deficient Transferrin as a Marker of Chronic Alcohol Abuse: A Critical Review of Preanalysis, Analysis, and Interpretation Clin. Chem., January 1, 2001; 47(1): 13 - 27. [Abstract] [Full Text] [PDF]
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