Transferrin isoelectric focusing for the investigation of congenital disorders of glycosylation: analysis of a ten‐year experience in a Brazilian center
Ana Paula Pereira Scholz de MagalhãesMaira Graeff BurinCarolina Fischinger Moura de SouzaFernanda BenderFernanda Medeiros SebastiãoThiago Oliveira SilvaFilippo Pinto e VairoIda Vanessa Döederlein Schwartz
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To characterize cases of suspected congenital disorders of glycosylation (CDG) investigated in a laboratory in southern Brazil using the transferrin isoelectric focusing TfIEF test from 2008 to 2017. Observational, cross‐sectional, retrospective study. The laboratory records of 1,546 individuals (median age = 36 months, 25‐75 IQR = 10‐108; males = 810) submitted to the TfIEF test during the period were reviewed. Fifty‐one individuals (3%) had an altered TfIEF pattern (5 ± 2.8 cases/year; median age = 24 months, 25‐75 IQR = 11‐57 months; males = 27, 53%). For 14 of them, data on diagnosis conclusion were available (classic galactosemia = 4; hereditary fructose intolerance = 4; peroxisomal diseases = 2; PMM2‐CDG = 2; MPDU1‐CDG = 1; SLC35A2‐CDG = 1).Comparing the cases with the normal and altered TfIEF patterns, there was a higher prevalence of altered cases in the age group from 11 months to 3 years. There was an increase in the likelihood of change in TfIEF, especially in the presence of inverted nipples or liver disease. The data suggest that the investigation of a case with suspected CDG is a complex problem, being aggravated by the existence of other IEMs (inborn errors of metabolism) associated with altered TfIEF pattern and lack of access to confirmatory tests. The presence of inverted nipples and liver disease, especially in individuals aged 11 months to 3 years, should suggest the need for TfIEF investigation. Caracterizar os casos com suspeita de CDG investigados em laboratório do sul do Brasil pelo exame de IEFTF de 2008 a 2017. Estudo observacional, transversal, retrospectivo. Foram revisadas as fichas laboratoriais de 1.546 indivíduos (mediana de idade = 36 meses, IQ 25‐75 = 10‐108; sexo masculino = 810) que fizeram o exame de IEFTF no período. Cinquenta e um indivíduos (3%) apresentaram padrão alterado na IEFTF (5 ± 2,8 casos/ano; mediana de idade = 24 meses, IQ 25‐75 = 11‐57 meses; sexo masculino = 27, 53%). Para 14 deles, estavam disponíveis dados sobre a conclusão do diagnóstico (galactosemia clássica = 4; intolerância hereditária à frutose = 4; doenças peroxissomais = 2; PMM2‐CDG = 2; MPDU1‐CDG = 1; SLC35A2‐CDG = 1). Comparando os casos com padrão normal e alterado na IEFTF, houve maior prevalência de casos alterados na faixa etária de 11 meses a 3 anos. Verificou‐se um aumento na probabilidade de alteração na IEFTF principalmente na presença de mamilos invertidos ou de hepatopatia. Os nossos dados sugerem que a investigação de um caso com suspeita de CDG é complexa, é agravada pela existência de outros EIM associados a padrão alterado na IEFTF e pela falta de acesso a exames confirmatórios. A presença principalmente de mamilos invertidos e de hepatopatia em indivíduos na faixa etária de 11 meses a 3 anos deve sugerir a necessidade de investigação por IEFTF.Keywords:
Carbohydrate deficient transferrin
Galactosemia
Liver disease
a personal density scanning imager (PDSI; Molecular Dynamics).Concentrations of LDL-chol and LDL-apo B were measured by a Hitachi 7170 automated analyzer (Hitachi), using Choletest LDL, and Apo B reagent kits (Daiichi Pure Chemical).These analyses were based on homogeneous enzymatic assay (8 ) and turbidimetric immunoassay (9 ) methods, respectively.The LDL-chol and LDL-apo B concentrations as well as the LDL particle sizes as measured by GGE and LDLchol/LDL-apo B ratios are summarized as the mean Ϯ SD in Table 1.A significant difference in LDL particle size was demonstrated between hyperlipemic and control samples by both GGE and the LDL-chol/LDL-apo B ratio.When LDL particle sizes measured by GGE were plotted against those determined in the same blood samples as LDL-chol/LDL-apo B ratios (Fig. 1), LDL-chol/LDL-apo B ratios showed a weak negative correlation with LDL particles sizes measured by GGE in control samples (Fig. 1A) and a weak positive correlation in hyperlipemic samples (Fig. 1B).In control samples, LDL-chol/LDL-apo B ratios were 1.14 -1.39, and LDL particle size measured by GGE was 21.38 -25.41 nm.Among hyperlipemic samples, despite reports that the LDL-chol/LDL-apo B ratio reflects the size of small dense LDL, some samples contained normal-sized LDL particles as determined by GGE but showed low LDL-chol/LDL-apo B ratios.This result reflects the fact that measurement of LDL-chol by the homogeneous enzyme assay is affected by high concentrations of TGs, which leads to underestimation of the LDL-chol concentration (8, 10 ).LDL size estimated by the LDL-chol/LDL-apo B ratio showed more negative correlation with TG concentrations than that estimated by GGE (r ϭ Ϫ0.68 vs Ϫ0.27).TG concentrations in these two samples were particularly high (8260 and 11 430 mg/L; Fig. 1B,Ⅺ).In contrast, some samples shown to contain small LDL particles by GGE showed high LDL-chol/LDLapo B ratios.The LDL-chol, LDL-apo B, and TG concentrations (Fig. 1B, ‚), respectively, were 1550, 1360, and 1810 mg/L in one sample and 1660, 1320, and 1890 mg/L in the other.When the LDL-chol/LDL-apo B ratio is used to evaluate LDL particle size, the size in these samples will be misreported as normal.The presence of small, dense LDL increases the risk of atherosclerotic cardiovascular disease beyond that associated with normal LDL (11 ).Given the importance of LDL particle size determination, it should be performed by GGE rather than rapidly estimated by the LDL-chol/LDLapo B ratio.
Carbohydrate deficient transferrin
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Measurement of an alcohol-induced shift in the serum transferrin glycosylation pattern, termed carbohydrate-deficient transferrin (CDT), is used as a biomarker for sustained high alcohol consumption. The present work examined whether non-enzymatic reaction of transferrin with glucose (glycation) might interfere with the use of CDT as an alcohol biomarker.The blood specimens were leftover volumes from the routine sample pool. Plasma and serum were collected among samples submitted for hemoglobin A1c (HbA1c) and CDT testing. Quantification of individual transferrin glycoforms in percentage of total transferrin was performed by an HPLC candidate CDT reference method.Incubating serum spiked with 20 or 200 mmol/l glucose caused time- and dose-dependent changes in the chromatographic profile of transferrin glycoforms, resulting in gradually wider peaks and reduced relative amounts of disialo- and trisialotransferrin. No similar chromatographic effects were seen in samples collected from diabetic patients with elevated HbA1c (>68 mmol/mol) values. These samples instead showed slightly higher mean %disialotransferrin levels (1.21%) compared with low HbA1c (<44 mmol/mol) samples (mean 1.06%; P = 0.023), pointing at a higher alcohol consumption level in the former group. Altogether ∼5% of the CDT values exceeded the cutoff. There was no significant difference in phosphatidylethanol (PEth) levels between the high and low HbA1c samples, but several (∼14%) showed elevated PEth concentrations.Glycation of serum transferrin in vivo was indicated to differ from that in vitro, and suggested not to interfere with %CDT testing by the HPLC method. The results indicated that CDT and PEth are useful as objective, complementary alcohol biomarkers to identify risky drinking also in diabetic subjects.
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CDT (carbohydrate-deficient transferrin) has been identified as a specific marker for chronically elevated alcohol consumption. We investigated the sensitivity and accuracy of using relative concentrations of different isotransferrins in serum for diagnosis of chronically elevated alcohol consumption. The different transferrin variants (isoforms) were quantified by HPLC. Including the trisialo-transferrin fraction into the definition of %CDT resulted in an increased accuracy in the detection of chronically elevated alcohol intake in a study among 17 heavy drinkers, 25 healthy individuals with moderate alcohol consumption and nine total abstainers. The results also suggest that desialylation of transferrin is a gradually continuing process, rather than one leading to a single end-result separating asialo-, mono- and disialo-transferrins from trisialo-, tetrasialo-, pentasialo- and higher sialo-transferrins.
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Elevated concentrations of carbohydrate‐deficient components of transferrin (CDT) in serum may be used as a sensitive and specific marker of regular, high alcohol consumption. When determined by a new, Simplified assay, CDT values are nearly normally distributed in low‐ or nonalcohol‐consuming control populations. The importance of transferrin phenotype for this normal variation was analyzed in 100 healthy, European men and women with no or negligible alcohol intake. No significant relation was found between phenotype and CDT value in this population. The three rare 6‐variants found had low CDT levels, and one subject, examined outside the study, with a rare D‐variant indicated that D‐variants may result in false‐positive CDT values. Moreover, women tended to have somewhat higher values than men, in whom CDT levels were weakly correlated with age. Other as yet undefined biological factors are clearly responsible for the major part of the normal variation of CDT values in nonalcoholic individuals.
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The introduction of a new set of reagents for the determination of carbohydrate-deficient transferrin (CDT) as a marker of chronic alcohol abuse requires an independent evaluation of the analytic specificity of the test. This information is needed for correct interpretation and classification of test results.Isoelectric focusing on the PhastSystem(TM) followed by immunofixation, silver staining, and densitometry was used to validate the initial transferrin isoform fractionation step on anion-exchange microcolumns involved in the ChronAlcoI.D. assay.The in vitro transferrin iron load was complete and stable. The CDT and non-CDT transferrin fractionation on anion-exchange microcolumns was reliable and reproducible (CV < or = 10%). Except for quantitatively unimportant traces of trisialo-Fe(2)-transferrin (<5% of total CDT), only asialo-, mono-, and disialo-Fe(2)-transferrin were detected in the microcolumn eluates (n = 170). There was a loss of proportionally similar amounts of asialo-Fe(2)-transferrin (during column rinsing) and disialo-Fe(2)-transferrin (on the anion exchanger). Thus, the peak height ratios for disialo- and asialo-Fe(2)-transferrin did not change from >1 (serum) to <1 (eluates) as described for the CDTect assays. The transferrin patterns in the ChronAlcoI.D. eluates were representative of those in serum. Transferrin D variants with isoelectric points close to that of trisialo-Fe(2)-transferrin C1 did not cause overdetermination of CDT by the ChronAlcoI.D. test.The initial CDT and non-CDT fractionation step involved in determination of CDT by the ChronAlcoI.D. assay is efficient for eliminating non-CDT transferrins from serum before quantification of CDT in the final turbidimetric immunoassay. We recommend IEF for validation of other (commercial) CDT analysis methods and of odd CDT results.
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Carbohydrate deficient transferrin
Linear relationship
Liver disease
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This chapter contains sections titled: Introduction Transferrin Microheterogeneity Carbohydrate-deficient Transferrin (CDT) Congenital Disorders of Glycosylation (CDG) Analytical Methods for Transferrin Microheterogeneity Chromatographic Methods for CDT Chromatographic Methods for CDG Summary and Conclusions References
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Carbohydrate-deficient transferrin (CDT) is widely accepted as screening test for excessive alcohol consumption. However, results from subjects with transferrin variants must be interpreted with caution since chromatography-based methods may give false-positive results. Furthermore, due to the co-elution in HPLC or the co-migration in capillary zone electrophoresis (CZE) of the di- and trisialylated C transferrins with the tetrasialylated D peak, exact measurement of CDT is impossible in CD-variants. Therefore, in this study, we tried to offer a different solution, including only the asialo-D, asialo-C, monosialo-D, monosialo-C, disialo-D and trisialo-D transferrins in the CDT calculation and referring to a different cut-off value for CDT in transferrin CD-variants. Comparison of alcohol consumers with teetotalers demonstrated area under the receiver operating characteristic curve of 0.79 and 0.76 for carbohydrate-deficient transferrin, 0.71 and 0.71 for mean corpuscular volume and 0.51 and 0.68 for gamma-glutamyltransferase in 43 subjects with transferrin CD-variants and 225 subjects with CC-phenotypes, respectively. Since false-positive carbohydrate-deficient transferrin results due to a transferrin CD-variant have major social implications, capillary electrophoresis-based or similar methods (HPLC, FPLC) should be preferred in populations carrying a high D-allele frequency.
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Abstract Background: Isoforms of transferrin interfere with measurement of carbohydrate-deficient transferrin (CDT) as a marker of heavy alcohol consumption. We evaluated the rate of inaccurate CDT results by immunoassays. Methods: We studied 2360 consecutive sera (1614 individuals) submitted for CDT assay without clinical information as well as samples from 1 patient with a congenital disorder of glycosylation (CDG Ia) and from 6 healthy carriers of CDG Ia. The CDTect, %CDT-TIA, and new %CDT immunoassays were compared with HPLC (%CDT-HPLC). Transferrin isoform pattern were evaluated by isoelectric focusing (IEF). Results: Transferrin BC and CD heterozygotes were found at frequencies of ∼0.7% and ∼0.2%, respectively. Another transferrin C subtype, where di- and trisialotransferrin partly coeluted (tentatively identified as C2C3), was observed in ∼0.6%. Compared with the %CDT-HPLC method, the immunoassays often produced low results for transferrin BC and high results for transferrin CD and “C2C3”. A very high trisialotransferrin value (frequency ∼1%) often produced high CDT immunoassay results. In four of six healthy carriers of CDG Ia, a- and disialotransferrin were highly increased and the HPLC and IEF isoform patterns were indistinguishable from those in alcohol abuse. Conclusions: Rare transferrin isoform types and abnormal amounts of trisialotransferrin (total frequency ∼2–3%) may cause incorrect determination of CDT with immunoassays. The observed variants were readily identified by HPLC and IEF, which can be recommended for verification of CDT immunoassay results in doubtful cases. In healthy carriers of CDG Ia, CDT is high by all assays.
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