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Enzymes and Protein Markers |
1
Abteilung für Klinische Chemie und Zentrallaboratorium, Baldingerstr., and
2
Medizinisches Zentrum für Nervenheilkunde, Funktionsbereich Neurochemie, Rudolf-Bultmannstr. 8, Philipps-Universität, D-35033 Marburg, Germany.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specific chemical reactions or antibodies for analysis of CDT are not available so far. CDT is usually determined, therefore, after separation from more-highly sialylated non-CDT-isotransferrins (tri-, tetra-, ... , octasialo-transferrin) by chromatographic (16)(17)(18)(19)(20)(21) or electrophoretic (5)(22)(23)(24)(25) methods. To reduce the number of microheterogeneous transferrin forms and to preclude coelution or cofocusing of transferrins with different contents of sialic acid and iron but the same isoelectric point (e.g., disialo-Fe2-transferrin as the main CDT-isotransferrin and tetrasialo-Fe1-transferrin as the main non-CDT-isotransferrin), analysis is usually done after in vitro iron saturation of the transferrin. With this sample pretreatment, only Fe2-transferrins (and no Fe1- and Fe0-transferrins) are present in the serum sample.
Commercially available sets of reagents for determination of CDT use in vitro iron saturation of transferrin to eliminate Fe1- and Fe0-transferrins and anion-exchange microcolumns to fractionate (separate) the non-CDT- and the CDT-isotransferrins. Subsequent quantification of the CDT-isotransferrins in column effluxes or eluates is done by RIA [CDTect-RIA (Pharmacia & Upjohn), and %CDT (AXIS, Oslo, Norway)], enzyme immunoassay (CDTect-EIA; Pharmacia & Upjohn), or turbidimetrically (CDTri-TIA; AXIS). Because anti-transferrin (and not anti-CDT) antibodies are used in these assays, the presence of non-CDT-isotransferrins in the column effluxes can lead to distinct overestimation of CDT. No quality-control material or external quality-control program for analysis of CDT is available so far, despite the increasing use of CDT, e.g., in forensic and employment medicine (26)(27)(28).
A unique definition of CDT is still missing. Until recently, all commercially available methods for determination of CDT and nearly all of the studies published summarized asialo-, monosialo-, and disialo-Fe2-transferrin as CDT (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(21)(22)(23)(24)(25)(26)(27). Now, a set of reagents incorporating trisialotransferrin in determination of CDT (CDTri-TIA) has been commercially launched. The same company offers a second set of reagents that does not include trisialotransferrin (%CDT). The directions for use of CDTect and %CDT give no clear information as to whether trisialotransferrin is completely, in part, or not at all determined with the asialo-, monosialo-, and disialo-Fe2-transferrin. Consequently, comparison and interpretation of CDT values are more and more difficult for the clinical chemist, the clinician, and the jurist.
The aim of our study was to investigate a widely distributed commercially available set of reagents, CDTect- RIA, for reliability of the initial anion-exchange isotransferrin fractionation step involved in the determination of CDT. We analyzed the isotransferrin patterns of serum samples by isoelectric focusing after each intermediate processing step. We wanted to get information about (a) the completeness of the in vitro transferrin iron-saturation step of the CDTect assay, which is used to eliminate the Fe1- and Fe0-transferrins; (b) the stability of the transferrin iron load during the passage of the serum sample through the chromatographic column, so as to prevent coelution of non-CDT- and CDT-isotransferrins of different iron contents; (c) the efficiency of the initial isotransferrin fractionation step at the anion-exchange microcolumns, and the fate of trisialotransferrin; (d) the reproducibility of the initial isotransferrin fractionation step, and (e) possible effects of variations of serum protein and transferrin concentrations on the CDTect assay performance.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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serum samples
All procedures were in accordance with the Helsinki Declaration of
1975, as revised in 1983. Only surplus serum sample volumes from
routine employment and forensic investigations and from patients
undergoing alcohol withdrawal treatment were used. For all subjects, an
18-variable clinical laboratory profile was established. The
patients had no signs of bacterial infection or inflammation, and
no infusions had been given. Blood was drawn after an overnight fast
into sterile gel-tubes (Sarstedt) in which the gel barrier consisted of
an polymerized acrylic resin. After clotting at room temperature for 30
min, the blood samples were centrifuged at 2000g for 10 min
at 4 °C. To avoid contamination of the serum samples with
microorganisms, the serum was removed with disposable pipettes. The
serum was split into one aliquot for routine laboratory analysis and
another aliquot (surplus serum volume) for determination of CDT. The
surplus serum volumes were immediately transferred into sterile,
leakproof plastic containers (1.2-mL Nalgene Cryotubes System) and
stored at -70 °C. By this means, serum samples were frozen within
30 min after centrifugation. They were thawed only once, on the day of
analysis.
methods
Assay of serum CDT concentration.
The serum
concentration of CDT was determined by CDTect-RIA in accordance with
the manufacturer's instructions. In short, after in vitro iron
saturation of transferrin (mixing 50 µL of serum sample, 200 µL of
ferric citrate solution, and 1 mL of buffer) and adsorption of
isotransferrins with isoelectric point values <5.7
(non-CDT-isotransferrins) on the anion-exchange microcolumns, the
isotransferrins with isoelectric point >5.7 (CDT-isotransferrins) in
the column effluxes were determined by means of a competitive,
double-antibody immunoadsorbent assay.
Quality control for the whole CDTect assay was done in accordance with the Guidelines of the Federal German Medical Association. In each run, serum pool aliquots with CDT values near the upper reference limits (20 U/L for men and 30 U/L for women) and a control sample supplied with the set of reagents were used for internal quality control, being placed at the beginning and end of each run. Control and serum samples were analyzed in duplicate. Intra- and interassay variations for the entire CDTect assay were 10% and 17%, respectively. Quality-control data were documented on quality-control sheets. No external quality-control program was available.
For validation by isoelectric focusing of the initial isotransferrin fractionation step used in determination of CDT by CDTect-RIA, we retained 10-µL aliquots of the original column effluxes. Column eluates were obtained by eluting the columns with 0.5 mL of 2 mol/L NaCl solution.
Isoelectric focusing.
Qualitative isoelectric focusing
on PhastSystemTM (Pharmacia/LKB) was done as described
previously (22), as modified (29). In short,
polyacrylamide gels, pH 5–6 (total acrylamide content T = 5%,
cross-linker content C = 3%; 43 x 50 x 0.45 mm;
Pharmalyte 5–6® diluted 1:16), adhering to a plastic
support film (GelBondTM PAG film; Biozym-Diagnostik,
Hameln, Germany), were prepared by us. After prefocusing of the gels
(75 V · h), 1 µL of pretreated sample was applied with use of
Sample ApplicatorTM 8/1 (Pharmacia/LKB)
(22)(29). Separation was performed for 200
V · h.
Sample pretreatment for isoelectric focusing.
To achieve
optimal specificity and sensitivity for detection of
CDT-isotransferrins, we diluted the iron-saturated serum samples and
NaCl eluates to a transferrin concentration of 7.5 mg/L (final dilution
factor 200- to 500-fold, depending on the original transferrin
concentration of the sample). The column effluxes were analyzed in the
original state (dilution factor 25-fold, after the initial serum
transferrin iron-saturation step). By this means, distinctly different
intensities (Fig. 1
, left panel) and peak heights (Fig. 1
, right panel) for
CDT-isotransferrin bands were obtained for serum samples and eluates on
the one hand and for effluxes on the other.
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Immunofixation.
Immunofixation was carried out as
described elsewhere (22)(29). In short, gels
were covered with polyclonal antibodies against transferrin, covered
with a plastic foil equal to the size of the gel to achieve an even
distribution of the antibody solution, and incubated at room
temperature in a moist chamber. Specificity of the transferrin
antibodies was tested as described (22). Unprecipitated
proteins were removed by washing with 150 mmol/L sodium chloride
solution overnight.
Visualization and evaluation of the transferrin bands.
The isotransferrin bands were visualized by silver staining in the
PhastSystem Development UnitTM as described previously
(22)(29). Transferrin bands were identified by
comparison with identically treated isotransferrin preparations or by
parallel analysis of cerebrospinal fluid samples, showing asialo- to
hexasialo-Fe2-transferrin bands (lane CSF in
Figs. 1–3
).
Gels were air-dried and stored for documentation.
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Densitometric evaluation of the isotransferrin bands (Figs. 1
, right,
and
2) was done with an Elscript 400 densitometer (Hirschmann
Gerätebau, Unterhaching, Germany) at 523 nm, with a diaphragm
aperture of 0.13 mm.
Other assays.
Serum protein concentration was determined
by use of the biuret method with a Hitachi 747 analyzer and reagents
from Boehringer Mannheim. Serum transferrin concentration was measured
immunonephelometrically with a Behring Nephelometer Analyzer (BNA) and
reagents from Behring. Internal and external quality control was done
in accordance with the Guidelines of the Federal German Medical
Association.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, one must consider the following: In
adjusting the sensitivity of the isoelectric focusing for detection of
CDT-isotransferrins, we accepted an overload of the ~100-fold greater
concentration of tetrasialo-Fe2-transferrin fraction. The
intensity of the tetrasialo-Fe2-transferrin band thus does
not correlate with the amount of this isotransferrin, making
quantification of CDT or measurement of the CDT/transferrin ratio in
the isoelectric focusing gel impossible.
cdtect assay
In vitro transferrin iron saturation.
In the case of
incomplete in vitro iron saturation of transferrin, bands of
Fe0- and Fe1-transferrins should be detected by
isoelectric focusing but will disappear after addition of further
amounts of Fe3. We did not find bands of Fe0-
and Fe1-transferrins in the iron-saturated serum samples.
Furthermore, we compared the isoelectric focusing isotransferrin band
patterns of serum samples after the Fe3 treatment
according to the CDTect test instructions and after Fe3
treatment (22). Varying the Fe3 treatment did
not affect the number and position of the transferrin bands or the
isotransferrin peak-height ratios. Both Fe3-treatment
procedures yielded isoelectric focusing isotransferrin band patterns
similar to those in lanes A of Fig. 1
, i.e., di-, tri-, tetra- (main
fraction), penta-, and hexasialo-Fe2-transferrins for
healthy controls with normal alcohol intake (<40 g of ethanol per
day). For alcoholics, a stronger disialo-Fe2-transferrin
band and additional bands of monosialo- and
asialo-Fe2-transferrin were found, reflecting the effects
of increased alcohol consumption.
Transferrin iron-load stability during column
passag
e. If transferrin iron is lost during column
passage, bands of Fe0- and Fe1-transferrins
should be detected in the column effluxes. Moreover, these bands should
disappear after addition of further amounts of Fe3 to the
effluxes. Of 130 column effluxes, 117 showed isoelectric focusing
transferrin band patterns in which the only bands present were
Fe2-transferrins; no bands for Fe0- or
Fe1- transferrins and no smear were present (lanes B of
Fig. 1
). A smear can result by the overlapping of several transferrins
with different iron and sialic acid contents because of transferrin
iron loss (22) during passage through the anion-exchange
column.
For 13 column effluxes, we obtained isotransferrin band patterns
similar to that of lane C in Fig. 3
, with CDT-isotransferrin bands (asialo-, monosialo-, and
disialo-Fe2-transferrin) and additional more-cathodic
bands (indicated by the arrowheads). The cathodic bands disappeared
after addition of further amounts of Fe3 to these column
effluxes. At the same time, new bands of more-highly sialylated
isotransferrins, possibly cofocused with CDT-isotransferrins because of
incomplete transferrin iron saturation, did not appear. The resulting
isoelectric focusing patterns for isotransferrin bands were similar to
those of lanes B of Fig. 1
(left). These findings indicate a weak iron
loss from CDT-isotransferrins during column passage in 13 of 130
columns. In none of the effluxes did we find higher-sialylated
Fe0- or Fe1- transferrins (tetra-, penta-,
hexa-, hepta-, and octasialotransferrins). From this we conclude that
the transferrin iron saturation was complete and sufficiently stable,
allowing a reliable fractionation of non-CDT- and CDT-isotransferrins
at the anion-exchange microcolumns. Thus, with correct use of the
CDTect assay, we conclude that transferrin iron-load stability is
sufficient during the isotransferrin fractionation step.
Annotation.
Effluxes of expired columns, or of columns
that had been in contact with air for 1 week (inappropriately stored
surplus columns from the previous analysis runs), contained distinct
amounts of Fe0- or Fe1-transferrins and
higher-sialylated isotransferrins (e.g.,
tetrasialo-Fe2-transferrin), which caused CDT
overestimations in the final RIA procedure. The corresponding band
patterns (not shown) were similar to that in lane B of Fig. 3
.
Efficiency of the isotransferrin fractionation at the
anion-exchange microcolumns.
The efficiency of the adsorption of
higher-sialylated, non-CDT-isotransferrins at the anion-exchange
column, as well as the elution behavior of the CDT-isotransferrins, was
investigated by isoelectric focusing analysis of the original
microcolumn effluxes and of additional 2 mol/L NaCl eluates.
Higher-sialylated, non-CDT-isotransferrins, especially
tetrasialo-Fe2-transferrin as the main isotransferrin, were
found in only 2 of 130 column effluxes (lane B of Fig. 3
). However,
these 2 columns were already showing a distinctly decelerated elution
speed during column equilibration, attributable to clogged filters at
the bottom of the columns. Of 130 column effluxes, 117 contained traces
[lanes B of Fig. 1
(left), lanes 1–7 of Fig. 2
), 1 contained distinct
amounts (lane A of Fig. 3
), and 12 no amounts of
trisialo-Fe2-transferrin. These trisialotransferrin
amounts, however, were quantitatively less important (<10% of the
final CDT result) and were within the analytical imprecision of 10%
for the whole CDTect assay.
The recovery of the CDT-isotransferrins was qualitatively assessed by
eluting the CDTect microcolumns with 2 mol/L NaCl solution and
isoelectric focusing analysis of the eluates. Typical isoelectric
focusing isotransferrin band patterns of these eluates are shown in
lanes C of Fig. 1
(left). In all eluates we found higher-sialylated,
non-CDT-isotransferrins (tri-, tetra-, penta-, and
hexasialo-Fe2-transferrin), which were correctly
adsorbed on the anion-exchange column. In all eluates, however, we also
found disialo-Fe2-transferrin (the main CDT-isotransferrin)
and, for alcoholics, asialo-Fe2-transferrin (lanes C of
Fig. 1
, left). This indicates a partial retention of these
isotransferrins at the anion-exchange column and thus incomplete
recovery of CDT. Because of the different final dilution factors (see
Materials and Methods), the isotransferrin peak heights
(Fig. 1
, right) are comparable only between serum (lanes A) and eluate
(lanes C), but not between serum and efflux (lanes B) or between efflux
and eluate (lanes B and C). For the alcoholic (CDT = 153 U/L; Fig. 1
, right), peak-height ratios of
disialo-Fe2-transferrin/asialo-Fe2-transferrin
>1 were obtained for serum and eluate (lanes A and C) and <1 for the
efflux (lane B). This switch is because of a lesser retention of
asialo-Fe2-transferrin than of
disialo-Fe2-transferrin at the anion-exchange column.
Reproducibility of the initial isotransferrin fractionation
step.
The within-run reproducibility of the CDTect initial
isotransferrin fractionation step was tested by processing 7 times
serum samples with normal and increased CDT. Calculating the mean, SD,
and CV 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 an imprecision of 
10% (Fig. 2
),
which was very close to that of the whole CDTect assay (10%
intraassay).
Effects of various serum protein and transferrin
concentrations on microcolumn function.
Various serum protein and
transferrin concentrations were achieved by dilution of serum samples
of healthy controls and alcoholics with 150 mmol/L NaCl solution. The
resulting protein and transferrin concentrations were 32–81 g/L (mean
55 g/L; n = 14) and 1.1–3.9 g/L (mean 2.3 g/L; n = 14),
respectively. The recovery of CDT in the diluted serum samples ranged
from 87% to 116% (mean 100%, n = 7). The correlation equation
between calculated (x) and measured CDT (y) was
y = 1.0x - 0.2 (r = 0.994).
This indicates a sufficient stability of the microcolumn anion-exchange
reactions to low serum protein and transferrin concentrations, and a
sufficient linearity of CDT measurement over the clinically significant
serum concentration ranges of protein, transferrin, and CDT.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The main problem of CDT analysis arises from the fact that the isotransferrins show very similar structural and chemical properties and extremely different serum concentrations (1)(2)(3). Thus, the CDT-isotransferrins (usually <30 mg/L; asialo-, mono-, and disialo-Fe2-transferrin) occur within a matrix containing 2200–4400 mg/L of higher-sialylated, non-CDT-isotransferrins, especially tetrasialotransferrin. Reliable chromatographic or electrophoretic separation of non-CDT- and CDT-isotransferrins is complicated by identical or very similar isoelectric point values of isotransferrins that have different iron and sialic acid contents (minimal isoelectric point difference 0.1 pH units) and by the presence of genetic variants (4)(5). Analysis is therefore usually done after elimination of Fe0- and Fe1-transferrins by in vitro iron saturation of transferrin. As we showed previously by isoelectric focusing (22), transferrin iron loss can occur under nonoptimal focusing conditions, such that non-CDT- and CDT-isotransferrins are cofocused. This transferrin iron loss may also occur under nonoptimal conditions at anion-exchange microcolumns, which are widely used in (commercially available) tests for determination of CDT.
The isotransferrin patterns in the anion-exchange microcolumn effluxes of the commercially available CDTect assay are studied here the first time. The data obtained in this study provide evidence for (a) complete elimination of Fe0- and Fe1-transferrins in the in vitro transferrin iron saturation step, (b) sufficient transferrin iron-load stability during column passage, and (c) reliable retention of non-CDT-isotransferrins (especially tetrasialo-Fe2-transferrin). Most of the column effluxes contained, besides asialo-, monosialo-, and disialo- Fe2-transferrin, traces of trisialo-Fe2-transferrin, which by definition does not belong to CDT (4). Complete separation of trisialotransferrin from CDT by anion-exchange chromatography is, however, very difficult, owing to the very close isoelectric points of disialo-Fe2-transferrin and tetrasialo-Fe2-transferrin. In the original method of Stibler et al. (reviewed in (4)), all isotransferrins with isoelectric points >5.65 were analyzed. However, the stability of this microcolumn isotransferrin separation was insufficient, owing to the very low ionic strength of the elution buffer (4). In their modified method, which is the basis for CDTect, "mainly components with isoelectric point values >5.7, and only a small amount of the isotransferrin with pI 5.7" are measured (4). Our results, however, demonstrate that traces of trisialo-Fe2-transferrin (<10% of CDT) are also present in the column effluxes. These contaminations were within the analytical imprecision of the whole assay and did not significantly affect the final CDT result.
Recently, Heggli et al. (20) reported increased trisialo-Fe2-transferrin concentrations in serum samples of alcoholics. Including this isotransferrin in determination of CDT improved the "distinction between social drinkers and heavy drinkers" and the authors conclude: "Further investigations to establish the best clinically analytical composition of transferrin variants in clinical testing of long-term alcohol intake may be fruitful" (20). Until now, a final decision as to whether trisialotransferrin should be incorporated into CDT is not possible. Definitely, the manufacturer of sets of reagents for determination of CDT must inform the users which isotransferrins are being analyzed as CDT. This information is missing in, e.g., the package inserts of the CDTect and the %CDT assays.
Whether trisialotransferrin is analyzed together with asialo-, monosialo-, and disialotransferrin is not only of scientific interest but also has practical implications. Under ordinary conditions, trisialotransferrin represents ~9% of the total serum transferrin concentration, in comparison with the 3% of disialo-, monosialo-, and asialotransferrin combined (30). Thus, complete, partial, or no coelution of this isotransferrin strongly affects such items as the upper reference limits that indicate chronic alcohol abuse (31). It may also affect the diagnostic sensitivity and specificity.
Of 130 columns, only 1 showed extraordinary amounts of
trisialo-Fe2-transferrin eluted (lane A of Fig. 3
) and
only 2 showed tetrasialo-Fe2-transferrin as the main
non-CDT-isotransferrin (lane B of Fig. 3
). However, during column
equilibration, these columns already showed a reduced elution speed.
For such columns, increased CVs for duplicate measurements were
obtained. We suggest a visual check of the anion-exchange microcolumn
elution behavior during the initial isotransferrin fractionation step
when determining CDT by the CDTect method. Columns with abnormal
elution behavior bear the risk of CDT overestimation and must be sorted
out. Although the column quality was very uniform, as indicated by CV
<10% for the CDT-isotransferrin peak-height ratios in the column
effluxes (Fig. 2
), we suggest a duplicate analysis for each serum
sample. This would prevent incorrect determination of CDT and false
results owing to nonoptimal performance of a single column. Variations
of >10% for the duplicate CDT measurement should be validated by
repetition of the analysis or by isoelectric focusing as the comparison
method. In our experience and with correct use of the CDTect assay, the
risk of overestimation of CDT on account of analytical
nonspecificities, e.g., partial or complete coelution of
tetrasialo-Fe2-transferrin with the CDT-isotransferrins, is
low.
A point that needs further investigation is the incomplete and variable
recovery of the different CDT-isotransferrins in the column effluxes.
Given the total lack of negatively charged sialic acid residues for
asialo-Fe2-transferrin, we conclude this
isotransferrin showed the lowest affinity to the anion-exchange
microcolumn and the best recovery. Owing to 2 negatively charged sialic
acid residues, disialo-Fe2-transferrin had a stronger
affinity to the anion-exchange material, resulting in partial retention
at the microcolumn (lanes C of Fig. 1
) and reduced recovery in the
microcolumn effluxes. This may be critical, because this isotransferrin
represents the main fraction of CDT. For CDT results at the
gender-specific upper reference limits, this may cause falsely negative
results. Obviously, in adjusting the assay for the greatest specificity
(minimizing the risk of coelution of
tetrasialo-Fe2-transferrin with CDT-isotransferrins, which
would cause overestimations and falsely positive results), an
incomplete recovery of CDT (and in some cases falsely negative results
for real CDT concentrations at the gender-specific upper reference
limits) was accepted. Thus, modifying the original method of Stibler et
al. as described above resulted in lower upper reference limits for the
CDT values that indicate greater alcohol consumption (4).
Accordingly, it is important not to confuse CDT values and upper
reference limits for CDT that were obtained by different analysis
methods.
An incomplete CDT recovery could be accepted if the recovery is constant over the clinically important concentration range. This was tested by twofold (and in some cases sixfold; data not shown) dilution of serum samples from controls and alcoholics. The correlation coefficient of 0.994 between calculated and measured CDT points to a sufficient linearity of the CDT recovery in the clinically important serum concentration ranges of protein, transferrin, and CDT.
A serious drawback of the isotransferrin fractionation by anion-exchange reactions is its failure to detect genetic transferrin variants that can cause false-positive (transferrin-D) or false-negative (transferrin-B) results. Further studies are required for investigating the possible effects of genetic transferrin variants or transferrin phenotypes on CDTect performance. For a satisfactory evaluation of this problem, analysis of serum samples to which have been added various amounts of appropriate isotransferrin preparations (which are thus far hard to obtain) is necessary. This was beyond the scope of this study. Possibly, individualized reference values, as recently suggested by Borg et al. for long-term monitoring of alcohol-dependent patients (32), may be useful for improving the diagnostic specificity and sensitivity of CDT in the case of genetic transferrin variants.
In conclusion, with correct use, the initial isotransferrin fractionation step involved in determination of CDT by the CDTect method was efficient for elimination of non-CDT-isotransferrins. According to personal information from the manufacturer, the same microcolumns are used in the CDTect-RIA and CDT-EIA reagent sets. The results obtained in this study for the determination of CDT by the CDTect-RIA method should therefore also be valid for CDTect-EIA. We recommend a procedure similar to that described here for analytical evaluation of other appropriate CDT analytical methods. Isoelectric focusing should be used for detection of transferrin variants or transferrin phenotypes and for validation of unusual or unexpected results obtained by other procedures. The data presented here indicate an urgent need for standardization of CDT analysis.
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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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-glutamyltransferase as markers of heavy alcohol consumption: gender differences. Alcohol Clin Exp Res 1994;18:747-754.
[Web of Science][Medline]
[Order article via Infotrieve]
-GT, methanol, isopropanol and ethanol in forensic blood samples for assessment of chronic alcohol abuse. Lab Med 1994;18:143-153.
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B. Wuyts, J. R. Delanghe, I. Kasvosve, A. Wauters, H. Neels, and J. Janssens Determination of Carbohydrate-deficient Transferrin Using Capillary Zone Electrophoresis Clin. Chem., February 1, 2001; 47(2): 247 - 255. [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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K. Scouller, K. M. Conigrave, P. Macaskill, L. Irwig, and J. B. Whitfield Should We Use Carbohydrate-deficient Transferrin instead of {gamma}-Glutamyltransferase for Detecting Problem Drinkers? A Systematic Review and Metaanalysis Clin. Chem., December 1, 2000; 46(12): 1894 - 1902. [Abstract] [Full Text] [PDF]
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R. Hackler, T. Arndt, A. Helwig-Rolig, J. Kropf, A. Steinmetz, and J. R. Schaefer Investigation by Isoelectric Focusing of the Initial Carbohydrate-deficient Transferrin (CDT) and non-CDT Transferrin Isoform Fractionation Step Involved in Determination of CDT by the ChronAlcoI.D. Assay Clin. Chem., April 1, 2000; 46(4): 483 - 492. [Abstract] [Full Text] [PDF]
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H. Henry, F. Froehlich, R. Perret, J.-D. Tissot, B. Eilers-Messerli, D. Lavanchy, C. Dionisi-Vici, J.-J. Gonvers, and C. Bachmann Microheterogeneity of Serum Glycoproteins in Patients with Chronic Alcohol Abuse Compared with Carbohydrate-deficient Glycoprotein Syndrome Type I Clin. Chem., September 1, 1999; 45(9): 1408 - 1413. [Abstract] [Full Text] [PDF]
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A. Helander Absolute or Relative Measurement of Carbohydrate-deficient Transferrin in Serum? Experiences with Three Immunological Assays Clin. Chem., January 1, 1999; 45(1): 131 - 135. [Full Text] [PDF]
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J. B. Whitfield, L. M. Fletcher, T. L. Murphy, L. W. Powell, J. Halliday, A. C. Heath, and N. G. Martin Smoking, obesity, and hypertension alter the dose–response curve and test sensitivity of carbohydrate-deficient transferrin as a marker of alcohol intake Clin. Chem., December 1, 1998; 44(12): 2480 - 2489. [Abstract] [Full Text] [PDF]
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A. Helander, E. Vabo, K. Levin, and S. Borg Intra- and interindividual variability of carbohydrate-deficient transferrin, {gamma}-glutamyltransferase, and mean corpuscular volume in teetotalers Clin. Chem., October 1, 1998; 44(10): 2120 - 2125. [Abstract] [Full Text] [PDF]
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T. Arndt, R. Hackler, and P. Bean Evaluation of Carbohydrate-deficient Transferrin Dr. Bean responds: Clin. Chem., May 1, 1998; 44(5): 1069 - 1071. [Full Text] [PDF]
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