www.jss-journal.com
Page 1
Journal of Separation Science
Determination of genetic transferrin variants in human serum by high-resolution capillary zone electrophoresis
Jitka Caslavska1, Jeannine Joneli1, Ursula Wanzenried1, Jeannette Schiess1, Christian Lanz2, Wolfgang Thormann1
1) Clinical Pharmacology Laboratory, Institute for Infectious Diseases, University of Bern, Bern, Switzerland. 2) Laboratory of Phytopharmacology, Bioanalytics and Pharmacokinetics, Department of Clinical Research, University of Bern, Bern, Switzerland. Short title: Genetic transferrin variants determined by capillary electrophoresis Abbreviations: CDT, carbohydrate-deficient transferrin; Tf, transferrin Keywords: carbohydrate-deficient transferrin, genetic transferrin variant, capillary electrophoresis
* Author, to whom correspondence should be addressed: Professor Dr. Wolfgang Thormann Clinical Pharmacology Laboratory Institute for Infectious Diseases Murtenstrasse 35 CH-3010 Bern, Switzerland phone: +41 31 632 3288; fax: +41 31 632 4997 email: [email protected]
Received: 06-Mar-2014; Revised: 08-Apr-2014; Accepted: 08-Apr-2014 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jssc.201400243. This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 2
Journal of Separation Science
Abstract High-resolution capillary zone electrophoresis in the routine arena with stringent quality assurance is employed for the determination of carbohydrate-deficient transferrin in human serum. The assay comprises mixing of human serum with a FeIII-containing solution prior to analysis of the iron saturated mixture in a dynamically double coated capillary using a commercial buffer at alkaline pH. In contrast to other assays, it provides sufficient resolution for proper recognition of genetic transferrin variants. Analysis of 7290 patient sera revealed 166 isoform patterns that could be assigned to genetic variants, namely, 109 BC, 53 CD, 1 BD and 3 CC variants. Several subtypes of transferrin D can be distinguished as they have large enough differences in pI values. Subtypes of transferrin C and B cannot be resolved. However, analysis of the detection time ratios of tetrasialo isoforms of transferrin BC and transferrin CD variants revealed multimodal frequency histograms, indicating the presence of subtypes of transferrin C, B and D. The data gathered over 11 years demonstrate the robustness of the high-resolution capillary zone electrophoresis assay. This is the first account of a capillary zone electrophoresis based carbohydrate-deficient transferrin assay with a broad overview on transferrin isoform patterns associated with genetic transferrin variants.
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 3
Journal of Separation Science
1 Introduction Transferrin (Tf) is an iron transporting glycoprotein with two iron binding sites and two Nlinked carbohydrate chains which are composed of a bi- to tetraantennary carbohydrate chain containing N-acetylglucosamine, mannose, galactose and terminal sialic acid residues. Due to different numbers of end standing sialic acid residues (zero up to eight), human serum comprises Tf isoforms which differ in their isoelectric points (between 5.9 and about 5.0 after complete iron saturation). The major Tf isoform in humans comprises four sialic acid residues (tetrasialo-Tf) and has an isoelectric point of 5.4 (after complete iron saturation). Carbohydrate-deficient transferrin (CDT) encompasses Tf isoforms with zero up to two sialic acid residues in the carbohydrate side chains of the molecule and become elevated in relation to total Tf upon ingestion of high amounts of ethanol over an extensive period of time. Thus, monitoring of CDT as biomarker for chronic excessive alcohol intake is of high interest for clinical and forensic purposes [1-8]. Human Tf is known to show genetic polymorphism caused by the substitution of one or more amino acids in the primary structure of the protein and a higher incidence of some genetic variants has been reported in connection with certain diseases. Up to now, at least 38 genetic Tf variants could be identified and only few of these occur with a prevalence of > 1%. Transferrin variants can be divided into three main groups, Tf-B, Tf-C and Tf-D. Tf-C, the most common phenotype in all populations, encompasses 16 distinct subtypes with slightly different isoelectric points, Tf-C1 being that with the highest prevalence (> 95%) in Caucasians. Tf-B variants with lower pI values than Tf-C and thus more anodically migrating variants occur with lower frequencies, whereas genetic Tf-D variants having higher pI values (more cathodically migrating variants) than Tf-C are reported to be rare in Caucasians, but not in some other populations [1,2,6,9-11]. Furthermore, congenital disorders of glycosylation (CDG) are a group of rare recessive inherited diseases with severe neurological and/or systemic manifestations from early childhood. They are characterized by defects in the synthesis of the glycan moieties of glycoconjugates, including those of Tf, such that Tf isoform patterns of CDG patients are typically quite different than those of healthy individuals and alcohol abusers [6,12-16].
Monitoring CDT, identification of Tf variants and assessment of CDG require methods that are capable of resolving Tf isoforms. Tf isoform patterns in human serum can be determined using gel isoelectric focusing [1,2,9,11,17-20], HPLC [6,7,14,21-24] and CZE [13,25-39]. Among all techniques, focusing on gels with immobilized pH gradients provides highest This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 4
Journal of Separation Science
resolution and thus the possibility to resolve subtypes of Tf-C with slightly different isoelectric points [18,19]. Assays based upon HPLC and CZE have the advantage that Tf isoform patterns are visualized in an instrumental format. In addition to the monitoring of CDT and CDG in human serum, various HPLC [6,7,37,41] and CZE [27,29,30,32,33,35,4043] assays were used to recognize genetic variants. However, no systematic assessment of genetic variants of Tf with these assays was reported. Most of these assays cannot be employed to properly fulfill this task as they lack sufficient Tf isoform resolution. CZE in fused-silica capillaries which feature dynamic capillary coatings formed by commercially available reagents was found to provide the highest Tf isoform resolution thus far reached with an instrumental approach [32,33,35]. These high-resolution assays were developed in our laboratory, reported to be suitable to recognize genetic variants of Tf, and applied to routine CDT testing during the past 11 years. In that time period, 7290 patient sera were analyzed under stringent quality assurance [36] and 166 isoform patterns (2.28%) could be assigned to genetic variants. Sera exhibiting genetic variants, including those assigned to Tf-CD, Tf-BC, Tf-BD and Tf-CC, were sorted based on type such that the prevalence of each group could be calculated for the analyzed collection of patient sera. Furthermore, for Tf-BC and Tf-CD variants, detection time ratios of tetrasialo-Tf peaks were found to provide multimodal frequency histograms which manifest the occurrence of variant subtypes of Tf-C, Tf-B and Tf-D even for cases in which high-resolution CZE cannot resolve all Tf isoforms. To our knowledge, this represents the first comprehensive collection of isoform patterns of genetic Tf variants assessed by CZE or any other instrumental separation method.
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 5
Journal of Separation Science
2 Materials and methods 2.1 Reagents, samples and sample preparation Buffers and reagents of the CEofix CDT kits No. 10-004740 and No. 10-004760 for the quantification of CDT in human serum were from Analis (Suarlée, Belgium). Rabbit antihuman Tf antibody (titer: 2800 mg/L) was purchased from Dako (Glostrup, Denmark). The chemicals were of analytical grade if not stated otherwise. Patient sera were received from hospitals, physicians and clinical laboratories for the determination of CDT. Sera were stored at –20°C until use. CDT ClinCheck serum control level II from Recipe (Munich, Germany) and the serum of a healthy subject, who gave his consent, were used as control sera as described in Ref. [36]. For iron saturation of all samples prior to analysis, 60 L of a serum and 60 L of the FeIII-containing solution of the reagent kits (Analis) were combined and briefly mixed [32,33]. For immunosubtraction of transferrin, 80 L of serum was incubated with 160 L of anti-human Tf antibody in a polypropylene vial for 45 min at room temperature. After centrifugation at 8000 g and 4C for 20 min, 60 L of the supernatant were collected, combined with 30 L of the ferric solution, briefly vortex mixed and analyzed as described before [32,33,35]. 2.2 CZE Instrumentation and running conditions First, analyses were performed on a P/ACE MDQ capillary electrophoresis system (Beckman Coulter) using the Analis reagent kit No. 10-004740 according to Lanz et al. [32] (referred to as first assay in this paper). Thereafter, Analis reagents No. 10-004760 were used on the same instrument according to Joneli et al. [33] and later on with the PA800plus capillary electrophoresis system (Beckman Coulter) as described elsewhere [36] (referred to as second assay in this paper). In all cases, a 50 m id fused-silica capillary (Polymicro Technologies, Phoenix, AZ, USA) of 60.2 cm (50 cm to the detector) total length was used. Samples were analyzed at a capillary cartridge temperature of 30.0°C, an applied voltage of 20.0 kV and having a detection wavelength of 200 nm (interference filter). In the first assay, samples were introduced by applying a vacuum (negative pressure) of 0.3 psi (1 psi = 6894.76 Pa) for 9–10 s [32]. For the second assay, application was effected with 0.5 psi for 12 s followed by a brief dip of the inlet capillary end into a vial containing water [33]. 2.3 Statistical and graphical data analysis
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 6
Journal of Separation Science
Statistical evaluations and graphical presentations were performed with SigmaPlot 12.5 (Systat Software, San Jose, CA). Each set of data was subjected to the normality test and comparison of two groups of data was performed with the Kruskal–Wallis One Way Analysis of Variance on Ranks test. P = 0.05 was considered the minimum level of statistical significance. 3 Results and discussion 3.1 Patterns of genetic variants During the 11 year period in the routine arena with stringent quality assurance as described in [36], our laboratory analyzed 7290 patient sera for CDT. Inspection of these results revealed 166 patterns (2.28%) that could be assigned to genetic variants (Table 1). The sera stemmed from 130 patients. The monitored electropherograms of genetic variants are a composite of two Tf isoform distributions of similar abundance which are essentially equal to that of a homozygote (Figure 1A) but with a shift in detection time of each corresponding isoform (Figures 1B–1E). The magnitude of the shift can be large (Figure 1D) or small (Figure 1E) and is dependent on the variant as well as the subtype of the variant as was shown by isoelectric focusing [1,2,9,11,17-20]. 53 samples (0.73% of total samples analyzed) revealed Tf-CD patterns in which the second pattern has a cathodic shift compared to that of Tf-C and thus earlier detection times (for example see Figure 1B) and 109 samples (1.50% of total samples analyzed) were Tf-BC distributions with an anodic time shift of the Tf-B pattern (shift to the right; Figure 1C). One sample stemmed from a Tf-BD heterozygote (Figure 1D) and 3 samples showed an unidentified Tf-CC heterozygote pattern (Figure 1E). Immunosubtraction of Tf and reanalysis of the supernatant revealed that all major peaks in the electropherograms were Tf isoforms and not any unknown interferences (dashed line graphs in Figure 1). Identification of patterns is accomplished by comparison of detection times with those of a control serum and/or with spiking the unknown sample with a serum of a known variant and reanalysis of the mixture. These approaches are shown with the electropherograms presented in Figure 2, data which were generated with the patient sera of panels C to E of Figure 1 and a control serum of a healthy human volunteer. For the Tf-BC variant (Figure 2A), both approaches revealed that the firstly detected major peak contains Tf-4C. Thus, for this example, analysis of a mixture of patient and control sample would not be necessary for proper variant assignment. This is due to the fact, that detection times of isoforms are very
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 7
Journal of Separation Science
reproducible [32,33]. However, for the small shift example shown in Figure 2C, analysis of the mixture is required to identify the part of the double peak which corresponds with the Tf4C peak of the control sample. It was found that the firstly detected peak of the double peak comigrated with the Tf-4C peak of the control sample (center graph of Figure 2C). This was quite puzzling as most Tf-C subtypes should be detected before the most abundant Tf-C subtype, Tf-C1 [9,17-20,44], and suggests that the Tf-CC pattern found here is a rare phenotype. For the patient whose electropherogram is shown in Figures 1E and 2C, samples were drawn seven weeks apart and the two analyses revealed identical electropherograms which demonstrates the high pattern reproducibility of the assay (data not shown). No efforts were undertaken to identify the second Tf-4C peak (back side of double peak in Figure 2C). However, the unusual pattern of Figure 2C prompted us to investigate the Tf isoform pattern of the control sample. Analysis of this serum by a gel isoelectric focusing test which is used to identify CDG at the Children’s Hospital of the University of Zürich (Zürich, Switzerland) revealed double bands for each Tf isoform. These double bands compare well with those of Tf-C1C2 found in the literature [11]. The isoelectric focusing data suggest that the control serum most likely represents a Tf-C1C2 heterozygote isoform distribution, a pattern which is quite often found within the Swiss population (25.3% of 759 persons tested [44]). Thus, TfC1C2 patterns cannot be resolved with our CZE assays. Furthermore, the serum of Figure 1E cannot stem from a Tf-C1C2 heterozygote as according to Ref. [44] the prevalence of this phenotype should be much larger than was observed with the three Tf-CC samples in our collection (Table 1). For the Tf-BD sample (Figure 2B), analysis of the mixture with the control sample revealed the occurrence of the Tf-4C peak (second graph from the top). The same was true for mixtures with Tf-BC and Tf-CD variants (second and third graphs, respectively, from the bottom). Furthermore, the Tf-CD sample augmented the Tf-4D peak in comparison to that of Tf-4B whereas with the Tf-BC serum an increased Tf-4B peak was observed. It was interesting to find that the peak shape of Tf-4D in the mixture was comparable to that monitored with the patient sample, indicating that the patient sample and the Tf-CD sample used to prepare the mixture could contain the same subtype of Tf-D. The Tf-4B peak in the mixture, however, was broader compared to that in the pattern of the patients sample (compare Tf-4B peaks of the lowest two electropherograms of Figure 2B) which indicates that there are most likely different Tf-B subtypes in the patient sample and the Tf-BC sample used to prepare the mixture. No efforts were undertaken to identify these subtypes.
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 8
Journal of Separation Science
3.2 Tf-BC variants For evaluation of the monitored Tf-BC patterns, the detection time ratio between detection times of Tf-4B and Tf-4C was calculated. Analysis of the detection time ratios was performed separately for the data of the two assays, as resolution of Tf-isoforms was noted to be different [32,33]. Box plots of the detection time ratios for the Tf-BC variants listed in Table 1 are presented in Figure 3A. Higher values were obtained for the second assay and statistical analysis using the Kruskal–Wallis one way analysis of variance (ANOVA) on ranks test revealed a statistical difference (P < 0.001) between the two sets of data. For each group, mean and median values were found to be different (Figure 3A) and analysis of the data with the Shapiro–Wilk normality test revealed that the data vary significantly from the pattern of a normal distribution. Furthermore, the box plots revealed outliers that appear to form groups. This is particularly well seen for the 92 data of the second assay and is further visualized with the multimodal frequency histogram presented in Figure 3B. The data of the second assay presented in Figure 3 can be divided into four groups (I–IV), with the data below the whisker of the 10th percentile representing group I, those around the box group II, those close but above the whisker of the 90th percentile group III and the two data points with the highest detection time ratio group IV. The occurrence of these groups is a strong indication of different subtypes of Tf-C and/or Tf-B. Assuming that the largest group with 71 samples (group II) represents Tf-C1B heterozygotes, samples with increased detection time ratios could represent Tf-C2B (e.g. group III) or other Tf-CB heterozygotes (group IV) as most Tf-C subtypes would be detected somewhat earlier than Tf-C1 as they have higher pI values than Tf-C1 [9,17-20,44]. Furthermore, samples of a patient drawn and analyzed on separate occasions revealed data within the same group. As an example, group IV of Figure 3B comprises two samples of one patient which were drawn two months apart and revealed detection time ratios of 1.0271 and 1.0274. No efforts were undertaken to identify subtypes of Tf-C and possible subtypes of Tf-B in these samples. 3.3 Tf-CD variants For evaluation of the monitored Tf-CD patterns, the detection time ratio between detection times of Tf-4D and Tf-4C was calculated for the samples analyzed with the two assays. Box plots of the detection time ratios for the Tf-CD variants listed in Table 1 are presented in Figure 4A. Lower values were obtained for the second assay and statistical analysis using the Kruskal–Wallis one way ANOVA on ranks test revealed a statistical difference (P = 0.008)
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 9
Journal of Separation Science
between the two sets of data. For the data of the second assay (n=49), mean and median values were found to be different and analysis of the data with the Shapiro–Wilk normality test revealed that the data vary significantly from the pattern of a normal distribution. Furthermore, the box plots revealed outliers that appear to be within selected groups. This is similar to what was observed for the Tf-BC variants but differences of the detection time ratios between groups were found to be larger. This is well seen by comparing the frequency histograms of Figures 4B and 3B. The second assay data of Figure 4 can be divided into four groups (I–IV), which can be associated with characteristic electropherograms (Figure 5) and thus believed to comprise different Tf-D subtypes. This is best seen by comparing electropherograms with those of a control serum (Figure 5). Samples of a patient drawn and analyzed on separate occasions revealed data within the same group. As an example, group IV of Figure 4B comprises four samples with two being from the same patient drawn and analyzed eight months apart. These samples revealed detection time ratios of 0.9777 and 0.9784, indicating a great stability of the ratios. The corresponding electropherogram is presented in Figure 5A. No efforts were undertaken to identify subtypes of Tf-D. Furthermore, a frequency histogram produced by the samples of group III (n=43, insert in Figure 4B) revealed a rather broad pattern which could be associated with the occurrence of different Tf-C subtypes that cannot be resolved and identified with the CZE assay. It is important to note that the Tf pattern monitored for group IV looks at first glance very similar to that of a Tf-BC heterozygote (compare patterns of Figures 5A and 1C). Careful comparison of the data of a control serum or analyzing a mixture of the patient serum with that of the control serum, however, properly reveals the assignment of one of the major peaks to Tf-4C and the other peak to the genetic Tf variant. 3.4 Unclear patterns In addition to the 166 samples listed in Table 1, a few samples (less than 0.1% of the total analyses) revealed unclear Tf patterns. Three unusual Tf patterns are presented in Figure 6. In these sera, immunosubtraction analysis revealed that all major peaks were Tf (compare dashed line graphs with solid line graphs of patient sera). The isoform patterns, however, could not be unambiguously assigned to one of the genetic variants discussed above. All sera which could be assigned to genetic variants (Table 1) comprised isoform distributions which were about equal, i.e. about 50:50% distributions for each isoform (Figures 1, 2 and 5). No
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 10
Journal of Separation Science
other distributions were reported thus far. Unequal distributions could in principle occur, but appear to be rare. The electropherograms shown in Figure 6A could potentially be assigned to a Tf-CD variant in which the Tf-4D peak is comigrating with Tf-3C and Tf-4C is detected together with Tf-5D. This would be in analogy to the case shown in Figure 5A (group IV of Figure 4B) with an unequal distribution of the isoforms (about 25 and 75% for Tf-D and TfC, respectively). Furthermore, the data of panels B and C of Figure 6 could be rare Tf-BC variants which comprise two Tf-B subtypes. No further identification of these sera was undertaken. 4 Concluding remarks Data gathered over 11 years demonstrate the robustness of the high-resolution CZE assays which are based on the CEofix reagents. The data obtained revealed that identification of genetic variants by CZE is straightforward provided that the resolution of the Tf isoforms is sufficient and there is an isoform distribution which is about 50:50%. According to our experience, the latter aspect is given in most cases. Assignment of a variant can be done with spiking the unknown sample with a control serum and reanalysis of the mixture or by comparison of the patient electropherograms with that of the control serum. Furthermore, resolution of the assays employed is insufficient to separate the common subtypes of Tf-C, including Tf-C1 and Tf-C2, and also subtypes of Tf-B. However, subtypes of Tf-D can be distinguished as they have larger differences in pI values [17,18]. Analysis of the detection time ratios of tetrasialo-Tf isoforms of Tf-CD and Tf-BC variants revealed multimodal frequency histograms, indicating the presence of subtypes of Tf-C, Tf-B and Tf-D variants. Due to insufficient isoform resolution, no other commonly used CZE assay can resolve the Tf isoform patterns as is described in this paper [35]. High resolution is also required to determine CDT levels in sera of genetic variants. CDT is taken as disialo-Tf, or the sum of asiolo-Tf and disialo-Tf, in relation to total Tf. The determination of CDT of genetic variants can be tricky as both disialo-Tf peaks should be completely resolved from other Tf isoforms. This, however, is not always the case even in our high-resolution assay format ([33], see Figures 1 and 5). For conditions with one resolved disialo-Tf peak, CDT values can be estimated with the assumption that the distribution is 50:50 such that the CDT value can be taken as two-fold the disialo-Tf value. Work is currently under way in our laboratory to establish separation conditions that would allow proper monitoring of CDT in sera of genetic Tf variants.
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 11
Journal of Separation Science
Acknowledgements This work was supported by the Swiss National Science Foundation. The authors acknowledge Dr. P. Burda, Children’s Hospital of the University of Zürich, for the IEF analysis of the control sample.
Conflicts of interest The authors have declared no conflict of interest.
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 12
Journal of Separation Science
5 References [1] de Jong, G., van Ejik, H.G., Electrophoresis 1988, 9, 589-598. [2] Arndt, T., Clin. Chem. 2001, 47, 13-27. [3] Musshoff, F., J. Chromatogr. B 2002, 781, 457-480. [4] Niemelä, O., Clin. Chim. Acta 2007, 377, 39-49. [5] Caslavska, J., Thormann, W., J. Sep. Sci. 2013, 36, 75-95. [6] Helander, A., Eriksson, G., Stibler, H., Jeppsson, J.-O., Clin. Chem. 2001, 47, 12251233. [7] Helander, A., Husa, A., Jeppsson, J.O., Clin. Chem. 2003, 49, 1881-1890. [8] Jeppsson, J.O., Arndt, T., Schellenberg, F.,Wielders, J.P.M., Anton, R.F., Whitfield, J.B., Helander, A., Clin. Chem. Lab. Med. 2007, 45, 558-562. [9] Kamboh, M.I., Ferrell, R.E., Hum. Hered. 1987, 37, 65-81. [10] Ibraimov, A.I., Sachkova, L.M., Kurmanova, G.U., Aksenrod, E.I., Mirrakhimov, M.M., Hum. Hered. 1993, 43, 53-57. [11] Marklová, E., Albahri, Z., Vaníček, H., Dědek, P., Vališ, M., Kopáčová, M., Vávrová, V., J. Inherit. Metab. Dis. 2008, 31, 457-461. [12] Henri, H., Froehlich, F., Perret, R., Tissot, J.-D., Eilers-Messerli, B., Lavanchy, D., Dionisi-Vici, C., Gonvers, J.-J., Bachmann, C., Clin. Chem. 1999, 45, 1408-1413. [13] Carchon, H.A., Chevigné, R., Falmagne, J.-B., Jaeken, J., Clin. Chem. 2004, 50, 101111. [14] Helander, A., Bergström, J., Freeze, H.H., Clin. Chem. 2004, 50, 954-958. [15] Marklová, E., Albahri, Z., Clin. Chim. Acta 2007, 385, 6-20. [16] Jaeken, J., Ann. N.Y. Acad. Sci. 2010, 1214, 190-198. [17] Kühnl, P., Spielmann, W., Weber, W., Hum. Genet. 1979, 46, 83-87. [18] Görg, A., Weser, J., Westermeier, R., Postel, W., Weidinger, S., Patutschnick, W., Cleve, H., Hum. Genet. 1983, 64, 222-226. [19] Pascali, V.L., Dobosz, M., Destro-Bisol, G., D’Aloja E., Electrophoresis 1988, 9, 411417. [20] Kishi, K., Ikehara, Y., Yasuda, T., Mizuta, K., Sato, W., Forensic Sci.Int. 1990, 45, 225-230. [21] Jeppsson, J.-O., Kristensson, H., Fimiani, C., Clin. Chem. 1993, 39, 2115-2120. [22] Werle, E., Seitz, G.E., Kohl, B., Fiehn, W., Seitz, H.K., Alcohol Alcohol. 1997, 32, 71-77. [23] Helander, A., Bergström, J.P., Clin. Chim. Acta 2006, 371, 187-190. [24] Schellenberg, F., Mennetrey, L., Girre, C., Nalpas, B., Pagès, J.C., Alcohol Alcohol. 2008, 43, 569-576. [25] F. Crivellente, G. Fracasso, R. Valentini, G. Manetto, A.P. Riviera, F. Tagliaro, J. Chromatogr. B 2000, 739, 81-93. [26] Lanz, C., Kuhn, M., Bortolotti, F., Tagliaro, F., Thormann, W., J. Chromatogr. A 2002, 979, 43-57. [27] Wuyts, B., Delanghe, J.R., Kasvosve, I., Gordeuk, V.R., Gangaidzo, I.T., Gomo, Z.A.R., Clin. Chem. Lab. Med. 2001, 39, 937-943.
This article is protected by copyright. All rights reserved.
www.jss-journal.com
[28]
[29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42]
[43] [44]
Page 13
Journal of Separation Science
Legros, F.J., Nuyens, V., Minet, E., Emonts, P., Zouaoui Boudjeltia, K., Courbe, A., Ruelle, J.-L., Colicis, J., de L’Escaille, F., Henry, J.-P., Clin. Chem. 2002, 48, 21772186. Legros, F.J., Nuyens, V., Baudoux, M., Zouaoui Boudjeltia, K., Ruelle, J.-L., Colicis, J., Cantraine, F., Henry, J.-P., Clin. Chem. 2003, 49, 440-449. Lanz, C., Marti, U., Thormann, W., J. Chromatogr. A 2003, 1013, 131-147. Lanz, C., Thormann, W., Electrophoresis 2003, 24, 4272-4281. Lanz, C., Kuhn, M., Deiss, V., Thormann, W., Electrophoresis 2004, 25, 2309-2318. Joneli, J., Lanz, C., Thormann, W., J. Chromatogr. A 2006, 1130, 272-280. Schellenberg, F., Wielders, J.P.M., Chim. Acta 2010, 411, 1888-1893. Marti, U., Joneli, J., Caslavska, J., Thormann, W., J. Sep. Sci. 2008, 31, 3079-3087. Joneli, J., Wanzenried, U., Schiess, J., Lanz, C., Caslavska, J., Thormann, W., Electrophoresis 2013, 34, 1563-1571. Caslavska, J., Joneli, J., Wanzenried, U., Schiess, J., Thormann, W., J. Sep. Sci. 2012, 35, 3521-3528. Pascali, J.P., Bortolotti, F., Sorio, D., Ivanova, M., Palmbach, T.M., Tagliaro, F., Med. Sci. Law 2011, 51, 26-31. Kuroda, Y., Hamaguchi, R., Moriyama, K., Tanimoto, T., Haginaka, J., J. Pharm. Biomed. Anal. 2013, 76, 81-86. Quintana, E., Montero, R., Casado, M., Navarro-Sastre, A., Vilaseca, M.A., Briones, P., Artuch, R., J. Chromatogr. B 2009, 877, 2513-2518. de Wolf, H.K., Huijben, K., van Wijnen, M., de Metz, M., Wielders, J.P.M., Clin. Chim. Acta 2011, 412, 1683-1685. Kasvosve, I., Delanghe, J.R., Gomo, Z.A.R., Gangaidzo, I.T., Khumalo, H., Wuyts, B., Mvundura, E., Saungweme, T., Moyo, V.M., Boelaert, J.R., Gordeuk, V.R., Clin. Chem. 2000, 46, 1535-1539. Thorn, J., Guillemin, H., de l’Escaille, F., J. Mol. Biol. 2009, 28, 305-307. Scherz, R., Reber, B., Pflugshaupt, R., Bütler, R., Electrophoresis 1985, 6, 569-571.
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 14
Journal of Separation Science
Figure 1. Electropherograms obtained with patient sera of (A) an alcohol abuser with increased levels of asialo-Tf and disialo-Tf, (B) a Tf-CD heterozygote alcohol abuser with increased levels of asialo-Tf and disialo-Tf, (C) a Tf-BC heterozygote, (D) a Tf-BD heterozygote and (E) a Tf-CC heterozygote of unknown identity. The broken line graphs represent data obtained after immunosubtraction of Tf. All presented data were produced according to the assay conditions of Joneli et al. [33]. Key: 0: asialo-Tf, 2: disialo-Tf, 3: trisialo-Tf, 4: tetrasialo-Tf, 5: pentasialo-Tf, 6: hexasialo-Tf.
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 15
This article is protected by copyright. All rights reserved.
Journal of Separation Science
www.jss-journal.com
Page 16
Journal of Separation Science
Figure 2. Electropherograms obtained with patient samples of (A) the Tf-BC heterozygote of Figure 1C, (B) the Tf-BD heterozygote of Figure 1D and (C) the Tf-CC of Figure 1E (bottom graphs) together with electropherograms of a control sample of a human volunteer (top graphs, presented with the y scale offset), 1:1 v/v mixtures of patient and volunteer samples (second graphs from top) and 1:1 v/v mixtures of patient sample and Tf-CD or Tf-BC samples of other patients (additional graphs in panel B). All presented data were produced according to the assay conditions of Joneli et al. [33]. For clarity, only tetrasialo-Tf peaks are labeled in patterns showing heterozygotes. Key as in Figure 1.
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 17
Journal of Separation Science
Figure 3. Detection time ratio data of Tf-BC variants (Tf-4B/Tf-4C) depicted (A) as separate box plots for the data of first (n=17) and second (n=92) assay (see Table 1) and (B) as a frequency histogram of the data of the second assay (n=92). Box plots are drawn using the standard percentile method, with lower and upper limits of the boxes representing the 25th and 75th percentiles. The horizontal lines within the boxes are median (solid line) and mean (broken line) and the whiskers indicate the 10th and 90th percentiles. The frequency histogram is divided into 16 bins of equal width.
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 18
Journal of Separation Science
Figure 4. Detection time ratio data of Tf-CD variants (Tf-4D/Tf-4C) depicted (A) as separate box plots for the data from of first (n=4) and second (n=49) assay (see Table 1) and (B) as a frequency histogram of the data of the second assay (n=49). Box plots are drawn using the standard percentile method, with lower and upper limits of the boxes representing the 25th and 75th percentiles. The horizontal lines within the boxes are median (solid line) and mean (broken line) and the whiskers indicate the 10th and 90th percentiles. The frequency histogram of panel B is divided into 16 bins of equal width. The insert in panel B depicts a 14 bin histogram of the data of group III (n=43).
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 19
This article is protected by copyright. All rights reserved.
Journal of Separation Science
www.jss-journal.com
Page 20
Journal of Separation Science
Figure 5. Electropherograms showing subtypes of Tf-CD heterozygote patients (bottom graphs) together with those of ClinCheck serum control samples (upper graphs). The patient electropherograms shown in panels A, B, C and D are from samples of groups IV, III, II and I, respectively, of Figure 4B. Detection time ratios (Tf-4D/Tf-4C) are 0.9777, 0.9683, 0.9576 and 0.9428, respectively. The patient electropherogram of panel B is the same as that of Figure 1B. All presented data were produced according to the assay conditions of Joneli et al. [33]. Key as for Figure 1.
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 21
Journal of Separation Science
Figure 6. Electropherograms showing unusual Tf isoform patterns of three patient sera (solid line bottom graphs) together with those of ClinCheck serum control samples (upper graphs). The broken line graphs represent data obtained after immunosubtraction of Tf in the patient sera. All presented data were produced according to the assay conditions of Joneli et al. [33]. Key as for Figure 1.
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 22
Journal of Separation Science
Table 1: Numbers of genetic variants and number of patients a) Variant
CD BC BD CC
Total analyses 53 109 1 3
Number of genetic variants Total in% b) First assay c) Second assay d) 0.727 1.495 0.014 0.041
4 17 1 0
49 92 0 3
Number of patients e) 40 87 1 2
a) Genetic variants found during analysis of a total of 7290 patient sera under routine conditions within 11 years. b) Number of genetic variants in relation to total number of analyses. c) CZE assay according to Lanz et al. [32]. d) CZE assay according to Joneli et al. [33]. e) Number of patients whose sera were analyzed once or multiple times.
. This article is protected by copyright. All rights reserved.
Page 1
Journal of Separation Science
Determination of genetic transferrin variants in human serum by high-resolution capillary zone electrophoresis
Jitka Caslavska1, Jeannine Joneli1, Ursula Wanzenried1, Jeannette Schiess1, Christian Lanz2, Wolfgang Thormann1
1) Clinical Pharmacology Laboratory, Institute for Infectious Diseases, University of Bern, Bern, Switzerland. 2) Laboratory of Phytopharmacology, Bioanalytics and Pharmacokinetics, Department of Clinical Research, University of Bern, Bern, Switzerland. Short title: Genetic transferrin variants determined by capillary electrophoresis Abbreviations: CDT, carbohydrate-deficient transferrin; Tf, transferrin Keywords: carbohydrate-deficient transferrin, genetic transferrin variant, capillary electrophoresis
* Author, to whom correspondence should be addressed: Professor Dr. Wolfgang Thormann Clinical Pharmacology Laboratory Institute for Infectious Diseases Murtenstrasse 35 CH-3010 Bern, Switzerland phone: +41 31 632 3288; fax: +41 31 632 4997 email: [email protected]
Received: 06-Mar-2014; Revised: 08-Apr-2014; Accepted: 08-Apr-2014 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jssc.201400243. This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 2
Journal of Separation Science
Abstract High-resolution capillary zone electrophoresis in the routine arena with stringent quality assurance is employed for the determination of carbohydrate-deficient transferrin in human serum. The assay comprises mixing of human serum with a FeIII-containing solution prior to analysis of the iron saturated mixture in a dynamically double coated capillary using a commercial buffer at alkaline pH. In contrast to other assays, it provides sufficient resolution for proper recognition of genetic transferrin variants. Analysis of 7290 patient sera revealed 166 isoform patterns that could be assigned to genetic variants, namely, 109 BC, 53 CD, 1 BD and 3 CC variants. Several subtypes of transferrin D can be distinguished as they have large enough differences in pI values. Subtypes of transferrin C and B cannot be resolved. However, analysis of the detection time ratios of tetrasialo isoforms of transferrin BC and transferrin CD variants revealed multimodal frequency histograms, indicating the presence of subtypes of transferrin C, B and D. The data gathered over 11 years demonstrate the robustness of the high-resolution capillary zone electrophoresis assay. This is the first account of a capillary zone electrophoresis based carbohydrate-deficient transferrin assay with a broad overview on transferrin isoform patterns associated with genetic transferrin variants.
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 3
Journal of Separation Science
1 Introduction Transferrin (Tf) is an iron transporting glycoprotein with two iron binding sites and two Nlinked carbohydrate chains which are composed of a bi- to tetraantennary carbohydrate chain containing N-acetylglucosamine, mannose, galactose and terminal sialic acid residues. Due to different numbers of end standing sialic acid residues (zero up to eight), human serum comprises Tf isoforms which differ in their isoelectric points (between 5.9 and about 5.0 after complete iron saturation). The major Tf isoform in humans comprises four sialic acid residues (tetrasialo-Tf) and has an isoelectric point of 5.4 (after complete iron saturation). Carbohydrate-deficient transferrin (CDT) encompasses Tf isoforms with zero up to two sialic acid residues in the carbohydrate side chains of the molecule and become elevated in relation to total Tf upon ingestion of high amounts of ethanol over an extensive period of time. Thus, monitoring of CDT as biomarker for chronic excessive alcohol intake is of high interest for clinical and forensic purposes [1-8]. Human Tf is known to show genetic polymorphism caused by the substitution of one or more amino acids in the primary structure of the protein and a higher incidence of some genetic variants has been reported in connection with certain diseases. Up to now, at least 38 genetic Tf variants could be identified and only few of these occur with a prevalence of > 1%. Transferrin variants can be divided into three main groups, Tf-B, Tf-C and Tf-D. Tf-C, the most common phenotype in all populations, encompasses 16 distinct subtypes with slightly different isoelectric points, Tf-C1 being that with the highest prevalence (> 95%) in Caucasians. Tf-B variants with lower pI values than Tf-C and thus more anodically migrating variants occur with lower frequencies, whereas genetic Tf-D variants having higher pI values (more cathodically migrating variants) than Tf-C are reported to be rare in Caucasians, but not in some other populations [1,2,6,9-11]. Furthermore, congenital disorders of glycosylation (CDG) are a group of rare recessive inherited diseases with severe neurological and/or systemic manifestations from early childhood. They are characterized by defects in the synthesis of the glycan moieties of glycoconjugates, including those of Tf, such that Tf isoform patterns of CDG patients are typically quite different than those of healthy individuals and alcohol abusers [6,12-16].
Monitoring CDT, identification of Tf variants and assessment of CDG require methods that are capable of resolving Tf isoforms. Tf isoform patterns in human serum can be determined using gel isoelectric focusing [1,2,9,11,17-20], HPLC [6,7,14,21-24] and CZE [13,25-39]. Among all techniques, focusing on gels with immobilized pH gradients provides highest This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 4
Journal of Separation Science
resolution and thus the possibility to resolve subtypes of Tf-C with slightly different isoelectric points [18,19]. Assays based upon HPLC and CZE have the advantage that Tf isoform patterns are visualized in an instrumental format. In addition to the monitoring of CDT and CDG in human serum, various HPLC [6,7,37,41] and CZE [27,29,30,32,33,35,4043] assays were used to recognize genetic variants. However, no systematic assessment of genetic variants of Tf with these assays was reported. Most of these assays cannot be employed to properly fulfill this task as they lack sufficient Tf isoform resolution. CZE in fused-silica capillaries which feature dynamic capillary coatings formed by commercially available reagents was found to provide the highest Tf isoform resolution thus far reached with an instrumental approach [32,33,35]. These high-resolution assays were developed in our laboratory, reported to be suitable to recognize genetic variants of Tf, and applied to routine CDT testing during the past 11 years. In that time period, 7290 patient sera were analyzed under stringent quality assurance [36] and 166 isoform patterns (2.28%) could be assigned to genetic variants. Sera exhibiting genetic variants, including those assigned to Tf-CD, Tf-BC, Tf-BD and Tf-CC, were sorted based on type such that the prevalence of each group could be calculated for the analyzed collection of patient sera. Furthermore, for Tf-BC and Tf-CD variants, detection time ratios of tetrasialo-Tf peaks were found to provide multimodal frequency histograms which manifest the occurrence of variant subtypes of Tf-C, Tf-B and Tf-D even for cases in which high-resolution CZE cannot resolve all Tf isoforms. To our knowledge, this represents the first comprehensive collection of isoform patterns of genetic Tf variants assessed by CZE or any other instrumental separation method.
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 5
Journal of Separation Science
2 Materials and methods 2.1 Reagents, samples and sample preparation Buffers and reagents of the CEofix CDT kits No. 10-004740 and No. 10-004760 for the quantification of CDT in human serum were from Analis (Suarlée, Belgium). Rabbit antihuman Tf antibody (titer: 2800 mg/L) was purchased from Dako (Glostrup, Denmark). The chemicals were of analytical grade if not stated otherwise. Patient sera were received from hospitals, physicians and clinical laboratories for the determination of CDT. Sera were stored at –20°C until use. CDT ClinCheck serum control level II from Recipe (Munich, Germany) and the serum of a healthy subject, who gave his consent, were used as control sera as described in Ref. [36]. For iron saturation of all samples prior to analysis, 60 L of a serum and 60 L of the FeIII-containing solution of the reagent kits (Analis) were combined and briefly mixed [32,33]. For immunosubtraction of transferrin, 80 L of serum was incubated with 160 L of anti-human Tf antibody in a polypropylene vial for 45 min at room temperature. After centrifugation at 8000 g and 4C for 20 min, 60 L of the supernatant were collected, combined with 30 L of the ferric solution, briefly vortex mixed and analyzed as described before [32,33,35]. 2.2 CZE Instrumentation and running conditions First, analyses were performed on a P/ACE MDQ capillary electrophoresis system (Beckman Coulter) using the Analis reagent kit No. 10-004740 according to Lanz et al. [32] (referred to as first assay in this paper). Thereafter, Analis reagents No. 10-004760 were used on the same instrument according to Joneli et al. [33] and later on with the PA800plus capillary electrophoresis system (Beckman Coulter) as described elsewhere [36] (referred to as second assay in this paper). In all cases, a 50 m id fused-silica capillary (Polymicro Technologies, Phoenix, AZ, USA) of 60.2 cm (50 cm to the detector) total length was used. Samples were analyzed at a capillary cartridge temperature of 30.0°C, an applied voltage of 20.0 kV and having a detection wavelength of 200 nm (interference filter). In the first assay, samples were introduced by applying a vacuum (negative pressure) of 0.3 psi (1 psi = 6894.76 Pa) for 9–10 s [32]. For the second assay, application was effected with 0.5 psi for 12 s followed by a brief dip of the inlet capillary end into a vial containing water [33]. 2.3 Statistical and graphical data analysis
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 6
Journal of Separation Science
Statistical evaluations and graphical presentations were performed with SigmaPlot 12.5 (Systat Software, San Jose, CA). Each set of data was subjected to the normality test and comparison of two groups of data was performed with the Kruskal–Wallis One Way Analysis of Variance on Ranks test. P = 0.05 was considered the minimum level of statistical significance. 3 Results and discussion 3.1 Patterns of genetic variants During the 11 year period in the routine arena with stringent quality assurance as described in [36], our laboratory analyzed 7290 patient sera for CDT. Inspection of these results revealed 166 patterns (2.28%) that could be assigned to genetic variants (Table 1). The sera stemmed from 130 patients. The monitored electropherograms of genetic variants are a composite of two Tf isoform distributions of similar abundance which are essentially equal to that of a homozygote (Figure 1A) but with a shift in detection time of each corresponding isoform (Figures 1B–1E). The magnitude of the shift can be large (Figure 1D) or small (Figure 1E) and is dependent on the variant as well as the subtype of the variant as was shown by isoelectric focusing [1,2,9,11,17-20]. 53 samples (0.73% of total samples analyzed) revealed Tf-CD patterns in which the second pattern has a cathodic shift compared to that of Tf-C and thus earlier detection times (for example see Figure 1B) and 109 samples (1.50% of total samples analyzed) were Tf-BC distributions with an anodic time shift of the Tf-B pattern (shift to the right; Figure 1C). One sample stemmed from a Tf-BD heterozygote (Figure 1D) and 3 samples showed an unidentified Tf-CC heterozygote pattern (Figure 1E). Immunosubtraction of Tf and reanalysis of the supernatant revealed that all major peaks in the electropherograms were Tf isoforms and not any unknown interferences (dashed line graphs in Figure 1). Identification of patterns is accomplished by comparison of detection times with those of a control serum and/or with spiking the unknown sample with a serum of a known variant and reanalysis of the mixture. These approaches are shown with the electropherograms presented in Figure 2, data which were generated with the patient sera of panels C to E of Figure 1 and a control serum of a healthy human volunteer. For the Tf-BC variant (Figure 2A), both approaches revealed that the firstly detected major peak contains Tf-4C. Thus, for this example, analysis of a mixture of patient and control sample would not be necessary for proper variant assignment. This is due to the fact, that detection times of isoforms are very
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 7
Journal of Separation Science
reproducible [32,33]. However, for the small shift example shown in Figure 2C, analysis of the mixture is required to identify the part of the double peak which corresponds with the Tf4C peak of the control sample. It was found that the firstly detected peak of the double peak comigrated with the Tf-4C peak of the control sample (center graph of Figure 2C). This was quite puzzling as most Tf-C subtypes should be detected before the most abundant Tf-C subtype, Tf-C1 [9,17-20,44], and suggests that the Tf-CC pattern found here is a rare phenotype. For the patient whose electropherogram is shown in Figures 1E and 2C, samples were drawn seven weeks apart and the two analyses revealed identical electropherograms which demonstrates the high pattern reproducibility of the assay (data not shown). No efforts were undertaken to identify the second Tf-4C peak (back side of double peak in Figure 2C). However, the unusual pattern of Figure 2C prompted us to investigate the Tf isoform pattern of the control sample. Analysis of this serum by a gel isoelectric focusing test which is used to identify CDG at the Children’s Hospital of the University of Zürich (Zürich, Switzerland) revealed double bands for each Tf isoform. These double bands compare well with those of Tf-C1C2 found in the literature [11]. The isoelectric focusing data suggest that the control serum most likely represents a Tf-C1C2 heterozygote isoform distribution, a pattern which is quite often found within the Swiss population (25.3% of 759 persons tested [44]). Thus, TfC1C2 patterns cannot be resolved with our CZE assays. Furthermore, the serum of Figure 1E cannot stem from a Tf-C1C2 heterozygote as according to Ref. [44] the prevalence of this phenotype should be much larger than was observed with the three Tf-CC samples in our collection (Table 1). For the Tf-BD sample (Figure 2B), analysis of the mixture with the control sample revealed the occurrence of the Tf-4C peak (second graph from the top). The same was true for mixtures with Tf-BC and Tf-CD variants (second and third graphs, respectively, from the bottom). Furthermore, the Tf-CD sample augmented the Tf-4D peak in comparison to that of Tf-4B whereas with the Tf-BC serum an increased Tf-4B peak was observed. It was interesting to find that the peak shape of Tf-4D in the mixture was comparable to that monitored with the patient sample, indicating that the patient sample and the Tf-CD sample used to prepare the mixture could contain the same subtype of Tf-D. The Tf-4B peak in the mixture, however, was broader compared to that in the pattern of the patients sample (compare Tf-4B peaks of the lowest two electropherograms of Figure 2B) which indicates that there are most likely different Tf-B subtypes in the patient sample and the Tf-BC sample used to prepare the mixture. No efforts were undertaken to identify these subtypes.
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 8
Journal of Separation Science
3.2 Tf-BC variants For evaluation of the monitored Tf-BC patterns, the detection time ratio between detection times of Tf-4B and Tf-4C was calculated. Analysis of the detection time ratios was performed separately for the data of the two assays, as resolution of Tf-isoforms was noted to be different [32,33]. Box plots of the detection time ratios for the Tf-BC variants listed in Table 1 are presented in Figure 3A. Higher values were obtained for the second assay and statistical analysis using the Kruskal–Wallis one way analysis of variance (ANOVA) on ranks test revealed a statistical difference (P < 0.001) between the two sets of data. For each group, mean and median values were found to be different (Figure 3A) and analysis of the data with the Shapiro–Wilk normality test revealed that the data vary significantly from the pattern of a normal distribution. Furthermore, the box plots revealed outliers that appear to form groups. This is particularly well seen for the 92 data of the second assay and is further visualized with the multimodal frequency histogram presented in Figure 3B. The data of the second assay presented in Figure 3 can be divided into four groups (I–IV), with the data below the whisker of the 10th percentile representing group I, those around the box group II, those close but above the whisker of the 90th percentile group III and the two data points with the highest detection time ratio group IV. The occurrence of these groups is a strong indication of different subtypes of Tf-C and/or Tf-B. Assuming that the largest group with 71 samples (group II) represents Tf-C1B heterozygotes, samples with increased detection time ratios could represent Tf-C2B (e.g. group III) or other Tf-CB heterozygotes (group IV) as most Tf-C subtypes would be detected somewhat earlier than Tf-C1 as they have higher pI values than Tf-C1 [9,17-20,44]. Furthermore, samples of a patient drawn and analyzed on separate occasions revealed data within the same group. As an example, group IV of Figure 3B comprises two samples of one patient which were drawn two months apart and revealed detection time ratios of 1.0271 and 1.0274. No efforts were undertaken to identify subtypes of Tf-C and possible subtypes of Tf-B in these samples. 3.3 Tf-CD variants For evaluation of the monitored Tf-CD patterns, the detection time ratio between detection times of Tf-4D and Tf-4C was calculated for the samples analyzed with the two assays. Box plots of the detection time ratios for the Tf-CD variants listed in Table 1 are presented in Figure 4A. Lower values were obtained for the second assay and statistical analysis using the Kruskal–Wallis one way ANOVA on ranks test revealed a statistical difference (P = 0.008)
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 9
Journal of Separation Science
between the two sets of data. For the data of the second assay (n=49), mean and median values were found to be different and analysis of the data with the Shapiro–Wilk normality test revealed that the data vary significantly from the pattern of a normal distribution. Furthermore, the box plots revealed outliers that appear to be within selected groups. This is similar to what was observed for the Tf-BC variants but differences of the detection time ratios between groups were found to be larger. This is well seen by comparing the frequency histograms of Figures 4B and 3B. The second assay data of Figure 4 can be divided into four groups (I–IV), which can be associated with characteristic electropherograms (Figure 5) and thus believed to comprise different Tf-D subtypes. This is best seen by comparing electropherograms with those of a control serum (Figure 5). Samples of a patient drawn and analyzed on separate occasions revealed data within the same group. As an example, group IV of Figure 4B comprises four samples with two being from the same patient drawn and analyzed eight months apart. These samples revealed detection time ratios of 0.9777 and 0.9784, indicating a great stability of the ratios. The corresponding electropherogram is presented in Figure 5A. No efforts were undertaken to identify subtypes of Tf-D. Furthermore, a frequency histogram produced by the samples of group III (n=43, insert in Figure 4B) revealed a rather broad pattern which could be associated with the occurrence of different Tf-C subtypes that cannot be resolved and identified with the CZE assay. It is important to note that the Tf pattern monitored for group IV looks at first glance very similar to that of a Tf-BC heterozygote (compare patterns of Figures 5A and 1C). Careful comparison of the data of a control serum or analyzing a mixture of the patient serum with that of the control serum, however, properly reveals the assignment of one of the major peaks to Tf-4C and the other peak to the genetic Tf variant. 3.4 Unclear patterns In addition to the 166 samples listed in Table 1, a few samples (less than 0.1% of the total analyses) revealed unclear Tf patterns. Three unusual Tf patterns are presented in Figure 6. In these sera, immunosubtraction analysis revealed that all major peaks were Tf (compare dashed line graphs with solid line graphs of patient sera). The isoform patterns, however, could not be unambiguously assigned to one of the genetic variants discussed above. All sera which could be assigned to genetic variants (Table 1) comprised isoform distributions which were about equal, i.e. about 50:50% distributions for each isoform (Figures 1, 2 and 5). No
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 10
Journal of Separation Science
other distributions were reported thus far. Unequal distributions could in principle occur, but appear to be rare. The electropherograms shown in Figure 6A could potentially be assigned to a Tf-CD variant in which the Tf-4D peak is comigrating with Tf-3C and Tf-4C is detected together with Tf-5D. This would be in analogy to the case shown in Figure 5A (group IV of Figure 4B) with an unequal distribution of the isoforms (about 25 and 75% for Tf-D and TfC, respectively). Furthermore, the data of panels B and C of Figure 6 could be rare Tf-BC variants which comprise two Tf-B subtypes. No further identification of these sera was undertaken. 4 Concluding remarks Data gathered over 11 years demonstrate the robustness of the high-resolution CZE assays which are based on the CEofix reagents. The data obtained revealed that identification of genetic variants by CZE is straightforward provided that the resolution of the Tf isoforms is sufficient and there is an isoform distribution which is about 50:50%. According to our experience, the latter aspect is given in most cases. Assignment of a variant can be done with spiking the unknown sample with a control serum and reanalysis of the mixture or by comparison of the patient electropherograms with that of the control serum. Furthermore, resolution of the assays employed is insufficient to separate the common subtypes of Tf-C, including Tf-C1 and Tf-C2, and also subtypes of Tf-B. However, subtypes of Tf-D can be distinguished as they have larger differences in pI values [17,18]. Analysis of the detection time ratios of tetrasialo-Tf isoforms of Tf-CD and Tf-BC variants revealed multimodal frequency histograms, indicating the presence of subtypes of Tf-C, Tf-B and Tf-D variants. Due to insufficient isoform resolution, no other commonly used CZE assay can resolve the Tf isoform patterns as is described in this paper [35]. High resolution is also required to determine CDT levels in sera of genetic variants. CDT is taken as disialo-Tf, or the sum of asiolo-Tf and disialo-Tf, in relation to total Tf. The determination of CDT of genetic variants can be tricky as both disialo-Tf peaks should be completely resolved from other Tf isoforms. This, however, is not always the case even in our high-resolution assay format ([33], see Figures 1 and 5). For conditions with one resolved disialo-Tf peak, CDT values can be estimated with the assumption that the distribution is 50:50 such that the CDT value can be taken as two-fold the disialo-Tf value. Work is currently under way in our laboratory to establish separation conditions that would allow proper monitoring of CDT in sera of genetic Tf variants.
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 11
Journal of Separation Science
Acknowledgements This work was supported by the Swiss National Science Foundation. The authors acknowledge Dr. P. Burda, Children’s Hospital of the University of Zürich, for the IEF analysis of the control sample.
Conflicts of interest The authors have declared no conflict of interest.
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 12
Journal of Separation Science
5 References [1] de Jong, G., van Ejik, H.G., Electrophoresis 1988, 9, 589-598. [2] Arndt, T., Clin. Chem. 2001, 47, 13-27. [3] Musshoff, F., J. Chromatogr. B 2002, 781, 457-480. [4] Niemelä, O., Clin. Chim. Acta 2007, 377, 39-49. [5] Caslavska, J., Thormann, W., J. Sep. Sci. 2013, 36, 75-95. [6] Helander, A., Eriksson, G., Stibler, H., Jeppsson, J.-O., Clin. Chem. 2001, 47, 12251233. [7] Helander, A., Husa, A., Jeppsson, J.O., Clin. Chem. 2003, 49, 1881-1890. [8] Jeppsson, J.O., Arndt, T., Schellenberg, F.,Wielders, J.P.M., Anton, R.F., Whitfield, J.B., Helander, A., Clin. Chem. Lab. Med. 2007, 45, 558-562. [9] Kamboh, M.I., Ferrell, R.E., Hum. Hered. 1987, 37, 65-81. [10] Ibraimov, A.I., Sachkova, L.M., Kurmanova, G.U., Aksenrod, E.I., Mirrakhimov, M.M., Hum. Hered. 1993, 43, 53-57. [11] Marklová, E., Albahri, Z., Vaníček, H., Dědek, P., Vališ, M., Kopáčová, M., Vávrová, V., J. Inherit. Metab. Dis. 2008, 31, 457-461. [12] Henri, H., Froehlich, F., Perret, R., Tissot, J.-D., Eilers-Messerli, B., Lavanchy, D., Dionisi-Vici, C., Gonvers, J.-J., Bachmann, C., Clin. Chem. 1999, 45, 1408-1413. [13] Carchon, H.A., Chevigné, R., Falmagne, J.-B., Jaeken, J., Clin. Chem. 2004, 50, 101111. [14] Helander, A., Bergström, J., Freeze, H.H., Clin. Chem. 2004, 50, 954-958. [15] Marklová, E., Albahri, Z., Clin. Chim. Acta 2007, 385, 6-20. [16] Jaeken, J., Ann. N.Y. Acad. Sci. 2010, 1214, 190-198. [17] Kühnl, P., Spielmann, W., Weber, W., Hum. Genet. 1979, 46, 83-87. [18] Görg, A., Weser, J., Westermeier, R., Postel, W., Weidinger, S., Patutschnick, W., Cleve, H., Hum. Genet. 1983, 64, 222-226. [19] Pascali, V.L., Dobosz, M., Destro-Bisol, G., D’Aloja E., Electrophoresis 1988, 9, 411417. [20] Kishi, K., Ikehara, Y., Yasuda, T., Mizuta, K., Sato, W., Forensic Sci.Int. 1990, 45, 225-230. [21] Jeppsson, J.-O., Kristensson, H., Fimiani, C., Clin. Chem. 1993, 39, 2115-2120. [22] Werle, E., Seitz, G.E., Kohl, B., Fiehn, W., Seitz, H.K., Alcohol Alcohol. 1997, 32, 71-77. [23] Helander, A., Bergström, J.P., Clin. Chim. Acta 2006, 371, 187-190. [24] Schellenberg, F., Mennetrey, L., Girre, C., Nalpas, B., Pagès, J.C., Alcohol Alcohol. 2008, 43, 569-576. [25] F. Crivellente, G. Fracasso, R. Valentini, G. Manetto, A.P. Riviera, F. Tagliaro, J. Chromatogr. B 2000, 739, 81-93. [26] Lanz, C., Kuhn, M., Bortolotti, F., Tagliaro, F., Thormann, W., J. Chromatogr. A 2002, 979, 43-57. [27] Wuyts, B., Delanghe, J.R., Kasvosve, I., Gordeuk, V.R., Gangaidzo, I.T., Gomo, Z.A.R., Clin. Chem. Lab. Med. 2001, 39, 937-943.
This article is protected by copyright. All rights reserved.
www.jss-journal.com
[28]
[29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42]
[43] [44]
Page 13
Journal of Separation Science
Legros, F.J., Nuyens, V., Minet, E., Emonts, P., Zouaoui Boudjeltia, K., Courbe, A., Ruelle, J.-L., Colicis, J., de L’Escaille, F., Henry, J.-P., Clin. Chem. 2002, 48, 21772186. Legros, F.J., Nuyens, V., Baudoux, M., Zouaoui Boudjeltia, K., Ruelle, J.-L., Colicis, J., Cantraine, F., Henry, J.-P., Clin. Chem. 2003, 49, 440-449. Lanz, C., Marti, U., Thormann, W., J. Chromatogr. A 2003, 1013, 131-147. Lanz, C., Thormann, W., Electrophoresis 2003, 24, 4272-4281. Lanz, C., Kuhn, M., Deiss, V., Thormann, W., Electrophoresis 2004, 25, 2309-2318. Joneli, J., Lanz, C., Thormann, W., J. Chromatogr. A 2006, 1130, 272-280. Schellenberg, F., Wielders, J.P.M., Chim. Acta 2010, 411, 1888-1893. Marti, U., Joneli, J., Caslavska, J., Thormann, W., J. Sep. Sci. 2008, 31, 3079-3087. Joneli, J., Wanzenried, U., Schiess, J., Lanz, C., Caslavska, J., Thormann, W., Electrophoresis 2013, 34, 1563-1571. Caslavska, J., Joneli, J., Wanzenried, U., Schiess, J., Thormann, W., J. Sep. Sci. 2012, 35, 3521-3528. Pascali, J.P., Bortolotti, F., Sorio, D., Ivanova, M., Palmbach, T.M., Tagliaro, F., Med. Sci. Law 2011, 51, 26-31. Kuroda, Y., Hamaguchi, R., Moriyama, K., Tanimoto, T., Haginaka, J., J. Pharm. Biomed. Anal. 2013, 76, 81-86. Quintana, E., Montero, R., Casado, M., Navarro-Sastre, A., Vilaseca, M.A., Briones, P., Artuch, R., J. Chromatogr. B 2009, 877, 2513-2518. de Wolf, H.K., Huijben, K., van Wijnen, M., de Metz, M., Wielders, J.P.M., Clin. Chim. Acta 2011, 412, 1683-1685. Kasvosve, I., Delanghe, J.R., Gomo, Z.A.R., Gangaidzo, I.T., Khumalo, H., Wuyts, B., Mvundura, E., Saungweme, T., Moyo, V.M., Boelaert, J.R., Gordeuk, V.R., Clin. Chem. 2000, 46, 1535-1539. Thorn, J., Guillemin, H., de l’Escaille, F., J. Mol. Biol. 2009, 28, 305-307. Scherz, R., Reber, B., Pflugshaupt, R., Bütler, R., Electrophoresis 1985, 6, 569-571.
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 14
Journal of Separation Science
Figure 1. Electropherograms obtained with patient sera of (A) an alcohol abuser with increased levels of asialo-Tf and disialo-Tf, (B) a Tf-CD heterozygote alcohol abuser with increased levels of asialo-Tf and disialo-Tf, (C) a Tf-BC heterozygote, (D) a Tf-BD heterozygote and (E) a Tf-CC heterozygote of unknown identity. The broken line graphs represent data obtained after immunosubtraction of Tf. All presented data were produced according to the assay conditions of Joneli et al. [33]. Key: 0: asialo-Tf, 2: disialo-Tf, 3: trisialo-Tf, 4: tetrasialo-Tf, 5: pentasialo-Tf, 6: hexasialo-Tf.
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 15
This article is protected by copyright. All rights reserved.
Journal of Separation Science
www.jss-journal.com
Page 16
Journal of Separation Science
Figure 2. Electropherograms obtained with patient samples of (A) the Tf-BC heterozygote of Figure 1C, (B) the Tf-BD heterozygote of Figure 1D and (C) the Tf-CC of Figure 1E (bottom graphs) together with electropherograms of a control sample of a human volunteer (top graphs, presented with the y scale offset), 1:1 v/v mixtures of patient and volunteer samples (second graphs from top) and 1:1 v/v mixtures of patient sample and Tf-CD or Tf-BC samples of other patients (additional graphs in panel B). All presented data were produced according to the assay conditions of Joneli et al. [33]. For clarity, only tetrasialo-Tf peaks are labeled in patterns showing heterozygotes. Key as in Figure 1.
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 17
Journal of Separation Science
Figure 3. Detection time ratio data of Tf-BC variants (Tf-4B/Tf-4C) depicted (A) as separate box plots for the data of first (n=17) and second (n=92) assay (see Table 1) and (B) as a frequency histogram of the data of the second assay (n=92). Box plots are drawn using the standard percentile method, with lower and upper limits of the boxes representing the 25th and 75th percentiles. The horizontal lines within the boxes are median (solid line) and mean (broken line) and the whiskers indicate the 10th and 90th percentiles. The frequency histogram is divided into 16 bins of equal width.
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 18
Journal of Separation Science
Figure 4. Detection time ratio data of Tf-CD variants (Tf-4D/Tf-4C) depicted (A) as separate box plots for the data from of first (n=4) and second (n=49) assay (see Table 1) and (B) as a frequency histogram of the data of the second assay (n=49). Box plots are drawn using the standard percentile method, with lower and upper limits of the boxes representing the 25th and 75th percentiles. The horizontal lines within the boxes are median (solid line) and mean (broken line) and the whiskers indicate the 10th and 90th percentiles. The frequency histogram of panel B is divided into 16 bins of equal width. The insert in panel B depicts a 14 bin histogram of the data of group III (n=43).
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 19
This article is protected by copyright. All rights reserved.
Journal of Separation Science
www.jss-journal.com
Page 20
Journal of Separation Science
Figure 5. Electropherograms showing subtypes of Tf-CD heterozygote patients (bottom graphs) together with those of ClinCheck serum control samples (upper graphs). The patient electropherograms shown in panels A, B, C and D are from samples of groups IV, III, II and I, respectively, of Figure 4B. Detection time ratios (Tf-4D/Tf-4C) are 0.9777, 0.9683, 0.9576 and 0.9428, respectively. The patient electropherogram of panel B is the same as that of Figure 1B. All presented data were produced according to the assay conditions of Joneli et al. [33]. Key as for Figure 1.
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 21
Journal of Separation Science
Figure 6. Electropherograms showing unusual Tf isoform patterns of three patient sera (solid line bottom graphs) together with those of ClinCheck serum control samples (upper graphs). The broken line graphs represent data obtained after immunosubtraction of Tf in the patient sera. All presented data were produced according to the assay conditions of Joneli et al. [33]. Key as for Figure 1.
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 22
Journal of Separation Science
Table 1: Numbers of genetic variants and number of patients a) Variant
CD BC BD CC
Total analyses 53 109 1 3
Number of genetic variants Total in% b) First assay c) Second assay d) 0.727 1.495 0.014 0.041
4 17 1 0
49 92 0 3
Number of patients e) 40 87 1 2
a) Genetic variants found during analysis of a total of 7290 patient sera under routine conditions within 11 years. b) Number of genetic variants in relation to total number of analyses. c) CZE assay according to Lanz et al. [32]. d) CZE assay according to Joneli et al. [33]. e) Number of patients whose sera were analyzed once or multiple times.
. This article is protected by copyright. All rights reserved.
