Search

A 2-year-old boy presented with developmental delay and rapidly progressing spasticity of lower limbs. A serum transferrin isoform test was requested as a work-up for congenital disorders of glycosylation (CDG). Figure 1 shows his serum transferrin isoelectric focusing pattern. Fig. 1. Lanes A–C, isoelectric focusing without neuraminidase; lanes D–F, isoelectric focusing after in vitro treatment of serum with neuraminidase. Lane A, normal serum; lane B, CDG type 1 patient; lane C, this patient; lane D, this patient; lane …

Background: Currently, monoclonal immunoglobulins are identified and quantified from bands on electrophoretic gels. As an alternative, clonality might be determined by measuring the separate light chain types of each Ig class to allow numerical assessment of Ig′κ/Ig′λ ratios, analogous to free light chain κ/λ ratios.Methods: Using immunization, tolerization, and adsorption procedures, we prepared sheep antibodies against each of the 6 separate molecules, IgGκ, IgGλ, IgAκ, IgAλ, IgMκ, and IgMλ. Antibody targets comprised the junctional epitopes between the heavy chain and light chain domains. After purification, we assessed the antisera on a Siemens Dade-Behring BN™ II nephelometer for analytical quality and clinical utility.Results: High-avidity, specific antibodies allowed the production of automated nephelometric immunoassays for each Ig light chain type. Laboratory comparison with serum protein electrophoresis, using dilution experiments, showed lower analytical sensitivity for monoclonal IgG detection but similar or greater sensitivity for IgA and IgM, particularly when the monoclonal bands overlaid transferrin. Results obtained from typing of monoclonal proteins into IgG, A, or M types were comparable with results obtained by immunofixation-electrophoresis methods. Initial clinical studies, in multiple myeloma patients, indicated that Ig′κ/Ig′λ ratios were sometimes more sensitive than immunofixation electrophoresis, provided numerical results, and correlated with changes in disease.Conclusions: Immunoassays for intact Ig κ/λ pairs are possible and should assist in the management of patients with monoclonal gammopathies.

Background: Diagnoses of congenital disorders of glycosylation (CDG) are based on clinical suspicion and analysis of transferrin (Tf) isoforms. Here we present our experience of CDG screening in children with a suspected metabolic disease by determination of serum percentage of carbohydrate-deficient transferrin (%CDT) in tandem with isoelectric focusing (IEF) analysis of Tf and α1-antitrypsin (α1-AT).Methods: We performed approximately 8000 serum %CDT determinations using %CDT turbidimetric immunoassay (TIA). In selected samples, IEF analysis of Tf and α1-AT was carried out on an agarose gel (pH 4–8) using an electrophoresis unit. The isoforms were detected by Western blotting and visualized by color development. We performed neuraminidase digestion of serum to detect polymorphic variants of Tf.Results: We established a cutoff value for serum %CDT of 2.5% in our pediatric population. Sixty-five patients showed consistently high values of serum %CDT. In accordance with Tf and α1-AT IEF profiles, enzyme assays, and mutation analysis, we made the following diagnoses: 23 CDG-Ia, 1 CDG-Ib, and 1 conserved oligomeric Golgi 1 (COG-1) deficiency. In addition, we identified 13 CDG-Ix non Ia, non-Ib; 3 CDG-Ix; and 9 CDG-IIx cases, albeit requiring further characterization; 9 patients with a secondary cause of hypoglycosylation and 6 with a polymorphic Tf variant were also detected.Conclusion: The combined use of CDT immunoassay with IEF of Tf and α1-AT is a useful 1st-line screening tool for identifying CDG patients with an N-glycosylation defect. Additional molecular investigations must of course be carried out to determine the specific genetic disease.

In this issue of Clinical Chemistry , Henderson et al. present a new, elaborate method that uses mass spectrometry to analyze and quantify serum vitamin D binding protein (DBP)2 (1). This protein, initially discovered by its different electrophoretic mobility in various populations and therefore called group-specific component (GC), is used in population genetics and forensic medicine (2, 3). The genetic basis of the polymorphism is known to be 2 amino acid differences (aa 432 and 436) in the C domain of the protein, thereby generating 3 isoforms, GC1f, GC1s, and GC2, detectable by genetic analysis (single nucleotide polymorphisms RS7041 and RS4588). Because both alleles are codominant, this genetic diversity generates 3 homozygous and 3 heterozygous possibilities. A remarkably strange south-to-north gradient is evident: members of the original African population (and their descendants around the world) mainly express GC1f, whereas populations living further from the equator have gradually higher percentages of GC1s or GC2 alleles. Moreover, based on isoelectric focusing, >120 different types of DBP GCs have been identified, although the genetic or posttranslational origin of these isoforms remains largely unexplained. DBP is a glycoprotein with either 3 (GC1 isoforms) or 2 carbohydrate chains on some (2%–20%) of the circulating DBP molecules (4); it is a multifunctional protein probably belonging to the oldest member of the albumin gene family (1, 2). It has a very high affinity for actin and helps to depolymerize filamentous actin into globular actin when released in the bloodstream after cell damage (5). It is also the major binding protein for all vitamin D metabolites, with higher affinity for 25-hydroxyvitamin D \[25(OH)D\] (the prohormone) than for 1,25-dihydroxyvitamin D [1,25(OH)2D] (the vitamin D hormone). This is very similar to the situation for thyroid hormones, in which the T4 binds thyroxine-binding globulin much …

Background: In diabetes, protein function is altered by glycation, but the impact on the Fe3+ binding and antioxidant functions of transferrin (Tf) is largely unknown. The aim of the present study was to investigate the effects of glycation on the distribution of Fe3+ on the two Fe3+-binding sites of Tf.Methods: In vitro glycation of Tf was accomplished by preincubation with glucose for 14 days. Tf was loaded with Fe3+ compounds to achieve theoretical Tf Fe3+ saturations of 32%, 64%, and 96% (monitored by spectrophotometry). Fe3+-Tf isoforms were separated by isoelectric focusing.Results: Fe3+ binding was highest when Tf was incubated with Fe:nitrilotriacetic acid and reached a steady state overnight. Increasing the Fe3+ load led to a shift of isoform profile toward the diferric form (Fe2-Tf): in freshly prepared Tf, Fe2-Tf represented 6%, 30%, and 66% of all isoforms at 32%, 64%, and 96% theoretical Fe3+ saturation, respectively. Fe3+ was equally distributed to the monoferric Tf forms with Fe3+ bound to the amino (Fe1N-Tf) and carboxy termini (Fe1C-Tf). Glycation decreased binding of Fe3+ to Tf (monitored at 450 nm). At low theoretical Fe3+ saturation (32%), glycation increased the mean (SD) proportion of Fe2-Tf: 18 (3)% in the presence of 33.3 mmol/L glucose vs 12 (4)% with 0 mmol/L glucose (P = 0.01). In contrast, at 96% theoretical Fe3+ saturation, Fe2-Tf decreased linearly with increasing glycation (r = 0.97; P = 0.008). Preincubation, independent of glycation, favored the Fe1N-Tf isoform at 64% theoretical Fe3+ saturation [27 (0.7)% vs 23 (1.1)% of the Fe1C-Tf isoform; P = 0.009].Conclusions: Glycation impairs Fe3+ binding and affects Fe3+-Tf isoform distribution depending on concentration. The diagnostic implications of these results need further elucidation in clinical studies.

Congenital disorders of glycosylation (CDG), formerly named carbohydrate-deficient glycoprotein syndrome, are rare hereditary disorders caused by mutations in the genes coding for enzymes involved in the biosynthesis of glycoproteins and other glycoconjugates (1). The clinical characteristics are variable, but often include psychomotor, growth, and mental retardation from early childhood. CDG are the result of defects in the assembly and transfer (type I) or processing (type II) of the glycan moieties, and as a result, the carbohydrate chains are either completely missing (type I) or structurally abnormal (type II). CDG-Ia is the most frequent subtype, with ∼500 patients reported, whereas

Background: Results on sera and calibrators vary 1.4- to 2.3-fold among commercial human chorionic gonadotropin (hCG) assays. The relative contributions of calibrators, standards, hCG charge isoforms, and major structural variants to this variation have not been quantified. Methods: Purified hCG was separated by isoelectric focusing into four fractions with pI ranges of 3–4, 4–5, 5–6, and 6–7. These four fractions together with pure hCG, hyperglycosylated hCG, hCG β-subunit (hCGb), nicked hCG, and hCGb core fragment (hCGbcf) were tested in nine commonly used commercial serum assays for hCG. The compositions of pure hCG preparations, standards, and commercial hCG preparations were determined by immunoassay. Results: The three pure hCG preparations and the four hCG charge isoforms each showed parallel responses in the nine commercial hCG assays. Although wide variations were found in the detection of hCG structural variants by the nine assays (range for hyperglycosylated hCG, 468-1544 IU/L; for hCGb, 3187-5535 IU/L; for nicked hCG, 2736-4240 IU/L; and for hCGbcf, Conclusions: Intermethod differences in hCG results were not explained by changes in responses attributable to hCG charge isoforms or to hCG structural variants, but wide variation was observed in concentrations of hCG structural variants in calibrators and in detection of these structural variants. Differences in assay specificity and in composition of the calibrators are the most likely sources of between-method variation.

Background: Congenital disorders of glycosylation (CDG) are usually diagnosed by isoelectric focusing (IEF) of serum transferrin (Tf). The aim of this study was to evaluate capillary zone electrophoresis (CZE) as a diagnostic alternative to IEF. Methods: We performed 792 CZE analyses of Tf, using the CEofixTM-CDT (carbohydrate-deficient transferrin) assay. Peak identification was based on relative migration times (RMTs) to reduce migration variability. Results: Tf profiles comprised three main groups (A–C). Groups A and B were characterized by one or two dominant tetrasialo-Tf peaks, whereas group C showed a widely variable Tf isoform composition. Group A was composed of four subgroups: a major group with a typical Tf profile (considered as reference group), two minor groups with decreased or moderately increased trisialo-Tf isoform, and a group showing the presence of unknown compounds with RMTs similar to mono- and disialo-Tf. However, these compounds were absent on IEF. Group C contained all profiles from patients with confirmed as well as putative CDG. From the reference group, 99% confidence intervals were calculated for the RMTs of the Tf isoforms, and percentiles representing the Tf isoform distributions were defined. Conclusions: All patients with abnormal IEF results and confirmed CDG were identified by CZE; thus, this method can be used as a diagnostic alternative to IEF in a manner suitable for automation. Because whole serum is analyzed, it should be kept in mind that CZE profiles can show substances other than Tf.

Background: Some of the genetic variants of hemoglobin (Hb) and their chemically modified species are known to affect the measurement of Hb A1c. The purpose of this study was to characterize Hb species in the blood sample of a 74-year-old German male with an exceptionally low Hb A1c value.Methods: Hemolysates from the propositus and a healthy individual were analyzed by electrophoresis, cation-exchange HPLC, boronate affinity chromatography, and electrospray ionization-mass spectrometry (ESMS). Genomic DNA was amplified by PCR, and the sequencing was performed on an ABI 310 sequencer. Functional properties of Hb were determined by oxygen equilibrium studies and CO recombination kinetics after flash photodissociation. Glycohemoglobin species were synthesized by incubating hemolysates with glucose.Results: A novel, electrophoretically silent β chain, β5(A2)Pro→Ala or Hb Görwihl, was detected by cation-exchange HPLC. It accounted for ∼44% of the total Hb and had functional properties similar to those of normal Hb A and a mild degree of heat instability. During incubation with glucose, glycation of the β chains (assessed by ESMS) in the hemolysate of a healthy volunteer was twice as fast as in hemolysate from the propositus.Conclusions: The substitution β5(A2)Pro→Ala seems to affect neither the functional properties nor the heterotropic interactions of Hb, but slows glycation of the N-terminal valine by an unknown mechanism.

A 61-year-old man was being assessed for multiple sclerosis. His paired cerebrospinal fluid (CSF) and serum samples were sent to the clinical biochemistry laboratory for oligoclonal band analysis by CSF isoelectric focusing and IgG immunofixation (Fig. 1). Creutzfeldt–Jakob disease immunoassay panel with CSF 14-3-3, tau, and S100B proteins was negative. Fig. 1. Isoelectric focusing and IgG immunofixation of the patient's samples. Lane 1, …

A sample submitted for hemoglobin (Hb) evaluation on a 5-day-old premature male infant with intestinal perforation, intraventricular hemorrhage, anemia, and sepsis contained 61.1% Hb A, 23.7% Hb F, 2.1% Hb A2, and peaks of 8.2% and 5.2% in the P2 and P3 regions of the Bio-Rad Variant II HPLC β thalassemia assay (Fig. 1). Isoelectric focusing …

Background: Proteomic technology permits the investigation of genetic metabolic diseases at the level of protein expression. Changes in the expression, polypeptide structure, and posttranslational modification of individual proteins can be detected in complex mixtures of proteins. Methods: We used high-resolution two-dimensional polyacrylamide gel electrophoresis to separate isoforms of plasma proteins and detect abnormalities of mass and/or charge. We confirmed the identity of the separated proteins by in-gel digestion with proteases and N-glycanases and then analyzed the released peptides and glycans by matrix-assisted laser-desorption ionization–time-of-flight mass spectrometry. Results: Complete characterization of the polypeptide sequences and glycosylation of α1-antitrypsin isoforms was achieved in plasma from controls and from patients with three different known α1-antitrypsin deficiencies and congenital disorder of glycosylation type Ia. Conclusions: This study shows that proteomic techniques are a powerful and sensitive means of detecting changes in the amino acid sequence and abnormal posttranslational modifications of specific proteins in a complex biologic matrix.

Erythrocytosis, which is characterized by high hematocrit (packed cell volume), is a common condition causing complications attributable to hyperviscosity of blood. According to modern nomenclature, patients can be subdivided into one group of “apparent erythrocytosis” and one of “absolute erythrocytosis” (1). The absolute-erythrocytosis group contains both genetic disorders, acquired conditions such as polycythemia, and secondary reactions attributable to chronic hypoxemia or renal disease. The occurrence of hemoglobin (Hb) variants with increased oxygen affinity is a well-known cause of hereditary erythrocytosis, but in recent years interest has also been given to mutations in the erythropoietin receptor gene (2). Theoretically, functional studies of oxygen saturation curves would be the ideal method to screen for Hb variants that cause erythrocytosis, but such methods are not generally available and there are diverging opinions whether P 50 measurements of stored or shipped samples can be used (3)(4). Of the nearly 200 Hb variants with increased oxygen affinity described, fewer than one-half have been found in conjunction with significant erythrocytosis (3). Several of the variants are attributable to amino acid substitutions that do not affect the net charge, i.e., the substitutions are “silent” when investigation is performed by conventional electrophoretic techniques. In contrast, almost all Hb variants in this group can readily be found by mass spectrometry exploiting the change in molecular weight of the variant globin. Using electrospray mass spectrometry (ESMS), we have screened 70 consecutive samples from patients with unexplained erythrocytosis that had showed normal Hb pattern on isoelectric focusing (IEF) (5) and HPLC (6)(7) analysis. The investigation was approved by the Ethics Committee at Huddinge University Hospital. In three cases, we detected β-globin variants. This report illustrates how the information from electrophoretic methods and the knowledge of the wild-type globin gene sequence can facilitate the interpretation of mass spectral …

Background: Congenital disorders of glycosylation (CDG) are autosomal recessive disorders that produce increased serum carbohydrate-deficient transferrin (CDT) isoforms. Methods to resolve CDT from fully glycosylated transferrin (Trf) have been based on a neutral shift in the isoelectric focusing (IEF) pattern or on a reduction in the negative charge, allowing resolution by anion-exchange chromatography. Our purpose was to develop a method of resolution and relative quantification of Trf isoforms using online immunoaffinity liquid chromatography–mass spectrometry (LC-MS).Methods: Serum (25 μL) was diluted with 100 μL of water before application to an immunoaffinity column that sequestered Trf isoforms. Trf isoforms were eluted from the immunoaffinity column, concentrated on a C4 column, eluted from the C4 column, and introduced into the mass spectrometer. Analysis of the Trf isoforms was entirely automated and completed in Results: The LC-MS method demonstrated that the major abnormal Trf isoforms in CDG lack one complete oligosaccharide structure (mono-oligosaccharide) or both oligosaccharide structures (a-oligosaccharide), but not the sialic acids, as presumed on the basis of IEF methods. Calculation of relative ratios among three possible species (mono-/di-oligosaccharide and a-/di-oligosaccharide) is reproducible [mean intra- and interassay CVs were 9.3% (n = 10) and 10% (n = 5), respectively]. A reference range for patients Conclusions: Advantages of the LC-MS method include improved sensitivity, minimal sample preparation, and an analysis time of 100 samples analyzed in a single day. Moreover, the nature of the oligosaccharide defect in CDG is accurately reflected by mass resolution, and subtle oligosaccharide truncations may also be detected by this method.

Background: 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. Methods: Isoelectric focusing on the PhastSystemTM followed by immunofixation, silver staining, and densitometry was used to validate the initial transferrin isoform fractionation step on anion-exchange microcolumns involved in the ChronAlcoI.D.TM assay. Results: The in vitro transferrin iron load was complete and stable. The CDT and non-CDT transferrin fractionation on anion-exchange microcolumns was reliable and reproducible (CV ≤10%). Except for quantitatively unimportant traces of trisialo-Fe2-transferrin (1 (serum) to Conclusions: The initial CDT and non-CDT fractionation step involved in determination of CDT by the ChronAlcoI.D. assay is efficient for eliminating non-CDT transferrins from serum before quantification of CDT in the final turbidimetric immunoassay. We recommend IEF for validation of other (commercial) CDT analysis methods and of odd CDT results.

We report the observation of abnormal protein glycosylation in a patient with achondroplasia in whom known causes of impaired glycosylation could not be found. Automated isoelectric focusing (IEF) was performed in our laboratory for the investigation of glycosylation disturbances of transferrin (1)(2). All procedures were carried out in accordance with the Helsinki Declaration of 1975 as revised in 1996. We used surplus serum samples as controls, and surprisingly, in one of those, IEF revealed a moderate increase in disialotransferrin and a slight increase in asialotransferrin (Fig. 1⇓ , lane 1) compared with control serum (Fig. 1⇓ , lane 2). A similar IEF pattern has been described, e.g., in alcohol abuse (3) or in carbohydrate-deficient glycoprotein (CDG) syndrome (4). The patient’s isotransferrin pattern had been stable in different blood samples for more than 3 years. The hypoglycosylation of transferrin was confirmed by an increased carbohydrate-deficient transferrin (CDT) concentration of 44 units/L (upper reference limit, 20 units/L) determined by the CDTect assay (Pharmacia & Upjohn). Figure 1. Fe2-isotransferrin ( Fe 2 -Tf ) band patterns in serum and cerebrospinal fluid ( CSF ) obtained by automated IEF. CSF serves as reference for the localization of isotransferrin bands. Lane 1 , patient’s serum; lane 2 , control serum. Clinically, the patient was a 41-year-old man in good general health. His past history included well-controlled arterial …

Background: Central Middlesex Hospital, in northwest London, has screened neonates for hemoglobinopathies, using the established manual technique of isoelectric focusing (IEF) since 1989. Recently, this laboratory has faced a large increase in the number of samples tested per year. This study compared the detection of hemoglobin abnormalities between the existing manual IEF method and that of automated cation-exchange HPLC to determine the reliability of HPLC and whether an automated system would save time in the laboratory.Methods: Over a 15-month period, 25 750 blood samples, collected by heel prick onto filter paper, were tested using HPLC, and the results were compared with those obtained with IEF.Results: HPLC and IEF each identified 568 patients with FAS, 151 with FAC, 49 with FAD-Punjab, 23 with FS, 3 with FC, 6 with FSC, 5 with FE, and 1 with FD. IEF detected 62 patients with FAE, whereas HPLC detected 63. This additional FAE was observed on repeat IEF. One additional heterozygote detected by HPLC was initially not observed by IEF, but was detected on repeat IEF. HPLC detected all but six cases of Hb Barts observed by IEF. One double heterozygote and four heterozygotes were detected by IEF, but not by HPLC. The detection of hemoglobin variants expressed at low concentrations was comparable for the two methods, and carryover was not observed in routine analysis on HPLC.Conclusions: HPLC is a sensitive, efficient, and time-saving alternative to IEF for the neonatal screening of common hemoglobinopathies.

A wide variety of commercial methods are available to measure glycohemoglobin (gHb). These methods measure various species of gHb, e.g., total gHb, hemoglobin A1 (Hb A1), or Hb A1c. Some methods are based on charge differences between glycated and nonglycated hemoglobins (e.g., cation-exchange chromatography, electrophoresis, and isoelectric focusing), whereas boronate affinity methods depend upon the binding of the sugar groups on the hemoglobin molecule (1). Immunoassay of Hb A1c depends on the presence of an epitope that includes glucose and N-terminal amino acids of the β chain of hemoglobin. These methods usually measure Hb A1c as a percentage of total hemoglobin. Some immunoassay methods also measure Hb S1c or Hb C1c, whereas others do not (2). The National Glycohemoglobin Standardization Program (NGSP) was established to certify the various commercial methods so that they can be related to the candidate reference method used in the Diabetes Control and Complications Trial (DCCT) (3)(4). Recently, the effects of Hb AC and Hb AS on one gHb immunoassay method were assessed (5). Samples containing Hb C trait showed modestly higher results than the HPLC method used. Although hemoglobin variants are relatively rare in Caucasians of northern European descent, the African-American population has an 8% prevalence of Hb AS and a 3% prevalence of Hb AC (6)(7). In this study, we describe the influence of Hb AS and Hb AC on four DCCT-traceable gHb methods. A total of 129 samples were collected in evacuated tubes containing EDTA as an anticoagulant. Samples with hemoglobin variants were identified by comparison of retention times on the Diamat system (Bio-Rad Clinical Laboratories) to known retention times for Hb S and Hb C. Of the 129 samples collected, 47 were homozygous for Hb A, 39 were …

The analysis of human hemoglobins (Hbs) is of medical importance in a number of congenital defects. The hemoglobinopathies are grouped into defective variants of Hb, such as Hb S and >600 other variants, and thalassemias characterized by abnormal expression of the genes for normal globin chains (1)(2)(3). Alkaline electrophoresis, performed historically on cellulose acetate and currently on agarose, combined with citrate agar electrophoresis at acidic pH is still widely used (4)(5)(6)(7), but high-performance cation-exchange chromatography (HPCEC) offers superior resolution, speed, and automation (8)(9)(10). Capillary electrophoresis uses numerous separation principles and shares with HPLC the advantages of high resolution and automation, with on-line detection and direct quantification (11)(12)(13)(14)(15). Capillary isoelectric focusing on coated capillaries can be used to study Hb (16)(17)(18)(19)(20)(21), but is slower than HPCEC (21). The first reported assays based on capillary zone electrophoresis (CZE) gave poor resolution of Hbs or were not quantitative (22)(23)(24). The aim of the present work was to evaluate a rapid CZE assay with dynamic coating of the fused-silica at alkaline pH to detect abnormal Hbs and to quantify Hbs for the diagnosis of thalassemias, and at acidic pH to confirm the identity of Hb variants. Reagents were obtained from Analis. The alkaline kit A2 contained a hemolyzer, an initiator consisting of a polycation, and an arginine buffer (pH 8.8) containing a polyanion. The acidic kit A1 has been described elsewhere for Hb A1c measurement (25). Adult and newborn samples were collected in EDTA-containing tubes at the maternity unit of the Hopital Saint-Antoine (AP-HP, Paris, France) and received in the laboratory for Hb analysis. Whole blood samples …

Serum concentration of carbohydrate-deficient transferrin (CDT) is used for laboratory diagnosis of chronic alcohol abuse. Using isoelectric focusing for validation of the initial isotransferrin fractionation step involved in the determination of CDT by the CDTect assay, we found a complete in vitro iron saturation of transferrin and sufficient stability of the transferrin iron load during column passage; effective separation of non-CDT-isotransferrins and CDT-isotransferrins at the microcolumns; partial coelution of trisialo-Fe2-transferrin, which did not significantly affect CDT measurement; partial retention of CDT-isotransferrins, especially disialo-Fe2-transferrin, which may cause falsely negative results for CDT at the upper reference limits; good precision of the isotransferrin fractionation step; and no significant effects of low concentrations of serum protein and transferrin. We strongly urge standardization of CDT analysis and suggest isoelectric focusing for validation of CDT analysis methods and verification of odd results.

Screening for α1-antitrypsin deficiency (AATD) can be performed with dried blood spots (DBS) on filter paper, which allows quantification of AAT by immunonephelometry (1), identification of the AAT phenotype by isoelectric focusing(2)(3), and/or AAT genotyping (4). In most diagnostic flow-charts, important decisions often depend on the AAT concentration determined from DBS; however, current methods are imprecise, and results from DBS are not identical to those obtained using serum samples(4). We wished to develop a method for quantifying AAT in DBS. We collected 75 specimens from healthy blood donors and 74 from patients with suspected AATD. Samples were centrifuged for 8 min at 1620 g , and the serum or plasma, without manifest hemolysis, was separated and stored at −80 °C. Blood was spotted on filter paper with dashed-line 13-mm printed circles (Schleicher & Schuell Grade 903; lot. W-011) and air-dried. Cards stored in plastic bags at room temperature can be kept for at least 1 year. We measured AAT in serum or plasma by nephelometry (Array 360 System) with a polyclonal anti-human AAT goat antibody and calibrator (CAL2, assigned AAT …

The respective patterns of cerebrospinal fluid (CSF)-restricted oligoclonal IgG bands (OCBs) and assessment of their similarity in serum and CSF are important laboratory tools for evaluating immune-mediated inflammation of the central nervous system. OCB detection is the diagnostic laboratory tool of choice for multiple sclerosis (MS), in the initial, clinically isolated syndromes as well as in the primary progressive forms (1). The imperatives of diagnostic certainty and therapeutic timeliness have made this laboratory test mandatory. Isoelectric focusing (IEF) with IgG immunodetection is the recommended technique for OCB measurements (1)(2). Many factors (e.g., variability in gel conductivity, quality of carrier ampholytes, and electroendosmosis) make IEF inherently difficult to standardize. Notwithstanding this shortcoming, the results of previously published studies on interlaboratory variability in OCB detection indicate …

BACKGROUND The currently recommended technologies of HPLC and isoelectric focusing for newborn blood spot screening for sickle cell disease (SCD) identify both the disease and carrier states, resulting in large numbers of infants being followed up unnecessarily. Analysis of blood spot tryptic peptides performed by using tandem mass spectrometry (MS/MS) is an alternative technology to detect hemoglobin (Hb) variant disorders. METHODS We analyzed 2154 residual newborn blood spots and 675 newborn blood spots from infants with Hb variants by using MS/MS after trypsin digestion. Screening cutoffs were developed by using the ratio between the variant peptide–to–wild-type peptide abundance for HbS, C, DPunjab, OArab, Lepore, and E peptides. A postanalytical data analysis protocol was developed using these cutoffs to detect only the disease states of SCD and not to identify carrier states. A parallel study of 13 249 newborn blood spots from a high-prevalence SCD area were analyzed by both MS/MS and HPLC. RESULTS Screening cutoffs developed distinguished the infants with the disease states of SCD, infants who were carriers of SCD, and infants with normal Hb. In the parallel study no false-negative results were identified, and all clinically relevant cases were correctly identified using the MS/MS protocol. Unblinding the data revealed a total of 328 carrier infants that were successfully excluded by the protocol. CONCLUSIONS The screening protocol developed correctly identified infants with the disease states of SCD. Furthermore, large numbers of sickle cell carrier infants were successfully not identified, thereby avoiding unnecessary follow-up testing and referral for genetic counseling.

We have developed a new procedure for purifying prostate-specific antigen (PSA) from human seminal fluid. The method is based on ammonium sulfate precipitation, hydrophobic interaction chromatography, gel filtration, and anion-exchange chromatography. It can be completed within 2 days with a recovery of intact PSA of 30%. By anion-exchange chromatography, five isoforms of PSA (A, B, C, D, and E) can be separated. The major form (PSA-B) consists of the intact enzyme, as shown by the occurrence of only one band of 33 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or nonreducing conditions, and by amino acid sequencing, which reveals only one amino-terminal sequence corresponding to the reported amino-terminal sequence of intact PSA. The specific absorbance of 1 g/L PSA-B at 280 nm was 1.61, and 80% of the PSA-B formed a complex with alpha 1-antichymotrypsin, indicating that it is enzymatically active. Three cleaved forms of PSA with different nicking sites and low enzymatic activity were separated from intact PSA by ion-exchange chromatography. In addition, we isolated a glycosylation variant, PSA-A, which showed a higher isoelectric point (pI = 7.2) than PSA-B (pI = 6.9) but similar enzymatic activity; this form accounts for 5-10% of total PSA. After treatment with sialidase, PSA-A and B had the same isoelectric point value (pI = 7.7).

We here report the characteristics of two rare alpha-chain hemoglobin (Hb) variants. The variants were found during quantification of HbA1c by cation-exchange HPLC with the Diamat glycohemoglobin analyzer. They were further characterized by isoelectric focusing and PolyCAT A cation-exchange chromatography. The structure of the abnormal Hbs was established by amino acid analysis after separation of the globin chains by reversed-phase chromatography, digestion with trypsin, separation of the peptides by reversed-phase chromatography, and amino acid sequencing. These studies showed that the two variants were Hb Broussais [alpha 90 (FG2)Lys-->Asn] and Hb Cemenelum [alpha 92 (FG4)Arg-->Trp].

Distinct genetic variants of apolipoprotein (apo) AI have been shown to influence concentrations of high-density lipoprotein (HDL) cholesterol. The genetic polymorphism of apo AIV may modulate HDL-cholesterol, plasma triglycerides, and lipoprotein(a) concentrations. There is evidence for an antagonizing role of apo AII in reverse cholesterol transport. Since genetic polymorphisms and variants of these apolipoproteins are detectable by isoelectric focusing (IEF), we developed a rapid and easy automated method for IEF analysis of apos AI, AII, and AIV on self-made or commercially available gels, using the PhastSystem. Diluted plasma or serum samples (1 microL) are applied automatically onto the gel and IEF is carried out for 35-45 min. Afterwards, the apo A bands are precipitated by specific polyclonal antibodies and visualized by automated silver staining. This rapid procedure is suitable as a routine or screening method for IEF analysis of these major HDL apolipoproteins.

Capillary isoelectric focusing (cIEF) was used to identify and quantify major and minor hemoglobin (Hb) variants. Whole blood (approximately 10 microL required) hemolysate was analyzed with a commercial instrument equipped with a 50 microns (i.d.) x 27 cm coated capillary filled with 20 g/L ampholytes (pH 6-8) in 4 g/L methylcellulose (MC). Cathode and anode solutions were 20 mol/L NaOH and 100 mol/L H3PO4 in MC, respectively. Samples (approximately 40 nL) were applied via autosampler by low-pressure injection, focused for 3 min at 30 kV, and mobilized by simultaneous voltage and low pressure past the detector, where absorbance at 415 nm was analyzed by an automated data acquisition system. Blood from subjects with sickle cell trait, Hb S/C disease, and various beta-thalassemias were analyzed by cIEF in < 15 min. cIEF was used to separate Hb S from Hb D-Los Angeles. Assay precision determined with commercial controls gave CV < 2% for Hb A and S, and 1-11% for minor Hb variants A2, F, and A1c. Results obtained by cIEF for patients' samples agreed well with values determined by conventional assays (r2 > 0.95). The results demonstrate that cIEF is a rapid, sensitive, high-resolution automated method for routine quantitative clinical analysis of Hb variants.

In the diagnosis of alcohol abuse transferrin (Tf) allelic D variants generate false-positive test results for carbohydrate-deficient transferrin (CDT) as assessed by their electrophoretic migration patterns. The predominant Tf C1 allele encodes a protein for which the most prevalent isoform has a pI of 5.4, i.e., four sialic acids and two bound ion molecules. Carriers of allele D encode Tfs with different amino acid sequences, for which the pI is > 5.7, despite their identical iron and carbohydrate composition. We used isoelectric focusing, immunoblotting, and laser densitometry (IEF-IB-LD) to distinguish Tf D variants from CDT. Alcohol abusers carrying the D chi allele tested CDT+; D chi nondrinkers were CDT-. Although normal controls (< 15 g of alcohol per day for 7-10 consecutive days) carrying variants D1, D2, or D chi exhibited abnormal IEF banding patterns, they did not generate false-positive results for CDT. D3 variants expressed isoforms that migrate at the same pI as CDT bands. Thus, IEF-IB-LD yields a highly resolved banding pattern to distinguish most Tf D variants from CDT.

Carbohydrate-deficient transferrins (CDTs), naturally occurring glycosylated transferrin proteins, are reported to be increased in the serum of individuals who consume large quantities of alcohol (ethanol). We compared two methods for the separation and quantification of CDT, using the same alcohol-dependent patients and age-, gender-, and race-matched controls as sources of samples for both assays. There was good correlation (r = 0.89) between the microcolumn anion-exchange chromatography/RIA (MAEC/RIA) procedure and the isoelectric focusing, immunoblotting, and laser densitometry (IEF/IB/LD) procedure. Receiver operating characteristic analysis suggested that the IEF/IB/LD procedure would perform slightly better than MAEC/RIA for the overall population. However, both assays were much more sensitive for the detection of heavy alcohol consumption in men, compared with women. Alcohol consumption in the week prior to CDT measurement correlated only weakly with the concentrations measured with either assay.

The risks associated with sickle cell and other untreated hemoglobinopathies have led to mandatory neonatal screenings in 41 of the 50 states in the US (1). Preliminary screenings are often performed by electrophoresis at alkaline pH on cellulose acetate, with follow-up analysis of abnormal samples by acid electrophoresis on citrate agar (2). Alternatively, isoelectric focusing (IEF) or HPLC can be used in the screenings (3), but other techniques offer advantages. In the past decade, new mass spectrometry (MS) approaches, including matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, have been applied to the study of proteins (4). MALDI-TOF MS offers a potent means of analyzing constituent chains of hemoglobin A (HbA) directly from samples as small as a single red blood cell (5). Mass accuracy and resolution are sufficient to determine the presence of point mutations within chains differing in mass by as little as ∼20 Da. This ability allows detection of the β6Glu→Val variant (∼30 Da lower in molecular mass) during direct analysis of the intact chains. Moreover, the site of mutation can be determined by combining MALDI-TOF MS with specific enzymatic digestion, i.e., “mass mapping”. Thus, MALDI-TOF MS has the potential to be used in place of conventional approaches (6) for HbAS/HbS screening in the general population. To date, however, the mass spectrometric approaches have not been taken to the high-throughput mode necessary for efficient population screening, nor have they been applied to neonatal blood samples, which contain roughly equal portions of HbA and HbF. We report here progress in the development of a high-throughput mass spectrometric screening approach capable of screening the hemoglobin of neonates at rates of ∼100 samples/h. In a blind analysis, 96 neonate blood samples were prepared for both intact protein molecular mass determination and mass mapping using parallel robotics (96-well format) and analyzed …

Case Description An 85-year-old man was being assessed for multiple sclerosis. The patient's paired cerebrospinal fluid (CSF) and serum samples were sent to the clinical biochemistry laboratory for oligoclonal-band analysis by CSF isoelectric focusing/IgG immunofixation (Fig. 1). Fig. 1. Isoelectric focusing/IgG immunofixation of the patient's specimens. Lane 1, serum sample; lane 1′, corresponding CSF …

BACKGROUNDα-1-Antitrypsin (A1AT) deficiency results from a genetic disorder at 2 common loci. Diagnosis requires quantification of A1AT and subsequent identification of the specific variant. The current algorithm of laboratory testing for the diagnosis of A1AT deficiency uses a combination of quantification (nephelometry), genotyping, and/or phenotyping. We developed a multiple reaction monitoring liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of A1AT and identification of the 2 most common deficiency alleles present in 95% of the patients with A1AT deficiency.METHODSSerum samples (n = 40) were digested with trypsin, and appropriate 13C/15N-labeled standard peptides were added. We performed LC-MS/MS analysis with a 0.5- by 150-mm C18 column and H2O:acetonitrile:n-propanol:formic acid (A:98:1:1:0.2 and B:10:80:10:0.2; flow 12 μL/min) mobile phase in positive ion mode on a TSQ Quantum triple quadrupole MS system. We measured the A1AT concentration by comparison to a calibration curve and determined the phenotype by the presence or absence of variant peptides. We compared the results to the current phenotyping assay by isoelectric focusing (IEF) and the immunonephelometry quantitative assay.RESULTSFor A1AT allele detection, in 39 of 40 samples the LC-MS/MS results were identical to those obtained by IEF gel electrophoresis. The single discrepant result was rerun by IEF at a lower dilution, and the results were in concordance. The A1AT quantification by LC-MS/MS also compared favorably with nephelometry.CONCLUSIONSThe LC-MS/MS method correlates well with current phenotyping and nephelometric assays and has the potential to improve the laboratory diagnosis of genetic A1AT deficiency.

We assessed the effect of particular apolipoprotein (apo) E phenotypes, lipoprotein(a) [Lp(a)], and other lipoproteins on the development of dyslipoproteinemia in 450 patients with type I diabetes, ages 13-14 years. The control group consisted of 450 healthy school children of both sexes, ages 13-14 years. Both groups were found to be normolipidemic, but the concentration of Lp(a) was significantly (P < 0.05) higher in the diabetic children than in the control group. Apo E 3/2 and apo E 4/4 phenotypes were more frequent in the group of diabetics. Diabetics with the apo E 3/3 phenotype had higher concentrations of very-low-density lipoprotein (VLDL) and Lp(a), and lower concentrations of low-density lipoprotein (LDL) than the apo E 3/3 nondiabetics. For apo E 3/2 phenotypes, total cholesterol, LDL cholesterol, LDL, apo A-I, and Lp(a) concentrations were higher in the diabetic children than in the control group; for apo E 4/3 phenotypes, this was true for triglycerides and VLDL cholesterol. The distribution of Lp(a) lipoprotein concentrations between 0.01 and > 0.5 g/L indicated a more frequent occurrence of higher Lp(a) values in diabetic children than in the control group. Results of this study indicate that an increased concentration of Lp(a) lipoprotein and apo E 3/2 and apo E 4/3 phenotypes contribute to the expression of dyslipoproteinemia in type I diabetes in childhood.

We investigated the prevalence and characteristics of intestinal alkaline phosphatase (ALP; EC 3.1.3.1) identified in human serum by cellulose acetate electrophoresis in 8% of fasting serum samples from hospital patients (n = 500) and in 35% of fasting serum samples from patients with diabetes mellitus (n = 106; not differentiated between types 1 and 2). The intestinal ALP electrophoretic band was usually heterogeneous and contained two major subtypes of ALP. Isoelectric focusing of intestinal-ALP-positive serum treated with levamisole and neuraminidase (EC 3.2.1.18) revealed two distinct regions of enzymatic activity that comigrated with ALP extracted from small intestinal and colonic mucosa. Anodic intestinal ALP was resistant to treatment with levamisole and neuraminidase and comigrated with ALP from small intestinal mucosa. The more-cathodic intestinal ALP, which comigrated with ALP from colonic mucosa, was completely inhibited by levamisole and converted by neuraminidase to a species with a more basic pI than that of neuraminidase-digested tissue-nonspecific form. This component of intestinal ALP may be of vascular origin.

In this method for automatically running and staining isoelectric focusing (IEF) gels, pre-made dehydrated polyacrylamide gels were rehydrated before assays run with the PhastSystem (Pharmacia LKB Biotechnology). The typing of genetic variants of hemoglobin and alpha 1-antitrypsin in narrow pH gradients (pH 6.7-7.7 and 4.2-4.9, respectively) was simple, convenient, and reproducible. The clinically important variants of alpha 1-antitrypsin (ZZ and SZ) were identified from serum or dried blood on filter paper. The fast screening of abnormal hemoglobin samples (HbS) for cases in clinical medicine was easily performed. The total analysis time for the phenotyping with conventional protein staining was approximately 60 min.

Detection of hemoglobinopathies in newborns is critical for the identification of those infants in need of follow-up care [reviewed in Ref. (1)]. For example, infants homozygous for the sickle cell mutation are at greater risk for developing fatal pneumococcal infections and sepsis, which can be prevented by prophylactic antibiotic therapy. At present, the majority of newborn screening for hemoglobin (Hb) variants is done by electrophoresis, isoelectric focusing, or HPLC (2) using Hb extracted from dried blood spots. Nevertheless, detection of adult Hb variants often is complicated by the presence of fetal Hb in neonatal blood. Alternative approaches to these protein-based methodologies have been developed that directly detect the presence of hemoglobinopathy-associated mutations in newborn DNA (3), and some have been adapted to use blood spots (4)(5). Using the S and E mutations in the β-globin gene as examples, we have developed a multiplexed, high-throughput methodology that uses an array of allele-specific fluorescent beads and the Luminex100 analyzer (Luminex Corporation, Austin, TX; www.luminexcorp.com). This methodology distinguishes between the S and E alleles and their wild-type counterparts, HbA and non-E, of the β-globin gene in each specimen, making it possible to determine the genotype at each locus. The methodology is based on the principle that fluorescent microspheres with unique fluorescent profiles, called classifications, can be cross-linked to different analyte-specific reagents and used to create a fluorescence-based array capable of simultaneously assaying multiple analytes in each sample (6). The bead classifications were obtained separately from the Luminex Corporation with surface carboxyl groups for chemical cross-linking to different analyte-specific reagents, which in our studies were 5′-amino-modified oligodeoxynucleotides. As indicated above, each bead classification has a unique spectral address based on its 658 nm/712 nm emission …

The vitamin D-binding protein, also known as group-specific component (Gc) or Gc-globulin, is a 51.2-kDa polymorphic protein of the α2-macroglobulin fraction of human plasma (1). In human population, three common alleles ( Gc1f , Gc1s , and Gc2 ) and >120 rare variant alleles have been classified by isoelectric focusing (2). Gc1f and Gc1s contain sialic acid residues, whereas Gc2 does not (2). Gc-globulin may have an important role in the activation of macrophages and in osteoclast differentiation from monocytes and thus may control bone morphogenesis and remodeling (3)(4)(5)(6)(7). In humans, deglycosylation of Gc-globulin, involving stepwise removal of β-galactose and sialic acid from the trisaccharide, leaving N -acetyl-galactosamine (GalNAc), produces a potent macrophage-activating factor (Gc-MAF) (3). GalNAc is considered to have a crucial role in the macrophage-activating and osteoclast-differentiating functions of Gc-MAF because the removal of this sugar has been shown to be associated with the loss or impaired function of macrophages (8)(9). In osteopetrotic rat and mice models (6)(7) and in a single human study (4), indirect data suggested a defect in lysophospholipid-inducible Gc-MAF, although direct estimation of this factor in healthy and diseased states has not been performed. Gc-MAF estimation has been hampered by the lack of a suitable detection system for determining the sterically exposed GalNAc, which is critical for the activating properties of Gc-MAF. Because no standard preparation of Gc-MAF (as opposed to Gc-globulin) is available, its presence must be inferred from its properties. We here describe a hybrid sandwich lectin-ELISA for the measurement of the sterically exposed GalNAc (10)(11) generated in vitro from Gc-globulin in the plasma of healthy subjects. Blood was collected from 11 healthy subjects by venipuncture into EDTA sample tubes, and the plasma was separated within 5 min …

The detection of Bence Jones protein, an important part of the investigation of suspected myeloma, is most commonly done by agarose or cellulose nitrate electrophoresis followed by immunofixation. Bence Jones protein is recognized as single or multiple bands of one type of light chain. Unfortunately, improvements in sensitivity of these techniques (use of high-affinity antisera and higher resolution electrophoresis) frequently allow detection of multiple light chain bands in the urine of patients who do not have a B-cell dyscrasia. The bands are usually kappa, although they may be accompanied by lambda bands. This pattern may lead to the misdiagnosis of Bence Jones protein and oligoclonal light chain production in patients. Here we show that this pattern is produced by polyclonal light chains; it is present in the urine of all patients with a tubular proteinuria of any etiology and may be induced in healthy individuals by blocking their renal tubular protein reabsorption. Polyclonal light chains separate into monomers and dimers on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and into four major bands with many minor bands by isoelectric focusing. This difference in charge and possibly size results in the banding pattern seen on good-quality electrophoresis and immunofixation.

We describe a disorder in which low-density lipoprotein (LDL)-cholesterol and apolipoprotein B are in low concentration (0.47 mmol/L and 0.28 g/L, respectively) and chylomicrons are still present in plasma after an 18-h fast. The d less than 1.006 fraction was isolated by flotation ultracentrifugation and the apolipoproteins were analyzed by electrophoresis, immunoblotting with anti-apolipoprotein B-100 antiserum, and isoelectric focusing. In the d less than 1.006 fraction of the fasting serum, we found an apolipoprotein B form with the same apparent molecular mass as apolipoprotein B-48 and similar in amount to apolipoprotein B-100 (respective percentages, 46% and 54%). The monosialylated form of the apolipoprotein C-III was severely decreased. After an oral fat load, the repartition of the two species of apolipoprotein B did not change greatly (respective percentages, 60% and 40%), and the concentration of serum triglyceride increased only from 1.20 to 1.65 mmol/L.

The degree of substitution of protein-ligand conjugates can be determined from the change of the isoelectric point (pI) of the protein when ligand molecules are attached to its surface. Specifically, the pI values of conjugates with known degrees of substitution are obtained by isoelectric focusing electrophoresis and are used to generate a calibration curve that relates these two variables. The shape of the curve is sigmoidal and can be predicted by a theoretical model that takes into account the degree of substitution and the amino acid composition of the protein. By using such a calibration curve, one may estimate the degree of substitution of a given protein-ligand conjugate from its pI value. The applicability of the method is demonstrated with conjugates of pyridoxal 5-phosphate and avidin.

Background: Neonatal screening programs for sickle cell disease are now widespread in North American and European countries. Most programs apply isoelectric focusing or HPLC to detect hemoglobin variants. Because tandem mass spectrometry (MS/MS) is being used for screening of inherited metabolic disorders and allows protein identification, it was worth testing for hemoglobinopathy screening.Methods: We minimized sample preparation and analysis times by avoiding prior purification, derivatization, or separation. We developed a tryptic digestion methodology to screen for the main clinically important variants (Hb S, Hb C, and Hb E) and β-thalassemia. To ensure proper discrimination between homozygote and heterozygote variants, we selected 4 transitions with good signal intensities for each specific peptide and calculated variant/Hb A ratios for each. Method validation included intra- and interseries variability, carryover, and limit of detection. We also performed a comparative study with isoelectric focusing results on 2082 specimens.Results: Intraassay imprecision values (CVs) varied between 2.5% and 30.7%. Interassay CVs were between 6.3% and 23.6%. Carryover was Conclusions: MS/MS is a reliable method for hemoglobinopathy neonatal screening.

A simple procedure for phenotyping apolipoprotein (apo) E directly from plasma has been developed for use in the lipid clinic laboratory. In this new method, 10 microL of serum or plasma is pretreated with neuraminidase (EC 3.2.1.18), which removes the sialic acid residues from apo E and eliminates additional bands, thereby ensuring correct phenotype assignment. After a rapid delipidation step, the samples are focused in vertical polyacrylamide mini-slab gels and immunoblotted with a polyclonal goat anti-apo E antibody, followed by a Protein G-peroxidase conjugate. The accuracy of this method was confirmed by comparison with the established procedure of phenotyping by isoelectric focusing of delipidated very-low-density lipoprotein. In addition, sera from 203 subjects from Vancouver, selected without conscious bias, were used to determine the local distribution of the apo E alleles. We estimate that the relative frequencies of apo E alleles epsilon 2, epsilon 3, and epsilon 4 in this population are 0.086, 0.761, and 0.153, respectively. The speed and convenience of using minigels make this procedure ideal for clinical laboratory applications and large population studies.

We describe an improved method for the classification of alpha 1-antitrypsin variants by isoelectric focusing in agarose. Identification of the three Pi M subtypes can now be made by using a narrow-range carrier ampholyte (pH 4.2-4.9) and pretreating serum with dithioerythritol-iodoacetic acid to enhance band resolution. Phenotype results for two groups of Pi M homo- and heterozygotes are compared to illustrate the improved accuracy of the new method.

Background: Carbonylation is an irreversible oxidative modification of proteins that has been linked to various conditions of oxidative stress, aging, physiological disorders, and disease. Increased oxidative stress is thus also considered to play a role in the pathogenesis of age-related neurodegenerative disorders such as Alzheimer disease (AD). In addition, it has recently become evident that the response mechanisms to increased oxidative stress may depend on sex. Several oxidized carbonylated proteins have been identified in plasma and brain of AD patients by use of 2-dimensional oxyblotting.Methods: In this pilot study, we estimated the concentrations and carbonylation of the most abundant cerebrospinal fluid proteins in aging women and men, both AD patients suffering from mild dementia and individuals exhibiting no cognitive decline. Oxidized carbonylated proteins were analyzed with 2-dimensional multiplexed oxyblotting, mass spectrometry, and database searches.Results: Signals for β-trace, λ chain, and transthyretins were decreased in probable AD patients compared with controls. The only identified protein exhibiting an increased degree of carbonylation in AD patients was λ chain. The concentrations of proteins did not generally differ between men and women; however, vitamin D–binding protein, apolipoprotein A-I, and α-1-antitrypsin exhibited higher extents of carbonylation in men.Conclusions: None of the brain-specific proteins exhibited carbonylation changes in probable AD patients compared with age-matched neurological controls showing no cognitive decline. The carbonylation status of proteins differed between women and men. Two-dimensional multiplexed oxyblotting is applicable to study both the concentrations and carbonylation of cerebrospinal fluid proteins.

Human adult hemoglobin (Hb) consists of HbA (96% of the total), HbA2 (3%), and HbF (1%). HbA contains a number of subfractions, including HbA1a2, HbA1b, and HbA1c (glycosylated Hb). HbA1c has been used as a clinical marker for blood sugar control for the past one to two months. Inappropriately low or high HbA1c concentrations in comparison with blood glucose concentrations are caused by various conditions of Hb structure and metabolism; under such conditions, the use of HbA1c as a clinical marker cannot be warranted. Abnormally low HbA1c concentrations are usually encountered in patients with high turnover rates of Hb, whereas disproportionately high HbA1c concentrations are found under relatively rare conditions. Various hereditary Hb variants have been reported to cause disproportionately high and low HbA1c titers, as determined by chromatographic methods such as HPLC (1)(2)(3)(4)(5)(6). Recently, another simple and specific method for the quantitation of HbA1c, a latex immunoagglutination (LA) method that uses a monoclonal antibody against a glucose moiety at the β-chain N-terminal, has been developed and used in place of HPLC methods. However, the relevance of the LA method for measurement of HbA1c concentrations in cases with Hb variants has not been tested. We report a case of a Hb variant (Hb Niigata) (7) with inappropriately increased and decreased HbA1c measured with HPLC and LA, respectively. Thus, LA may have a limitation for the accurate quantitation of HbA1c in some special cases. A 58-year-old male (weight, 58.8 kg; height, 162.5 cm) without any abnormal signs or symptoms was evaluated with routine laboratory data at the time of an annual health check provided by his employer. His HbA1c measured with LA was below the reference interval (2.9%), but his fasting blood glucose (FBG) was 5.4 mmol/L (98 …

Leakage of cerebrospinal fluid (CSF) from the subarachnoid space into the nasal or aural cavity creates a pathway for life-threatening central nervous system infection. Detection of the leakage can be cumbersome (1). In former days the identification and localization of CSF leakage was almost entirely dependent upon the clinical history and methods involving intrathecal injection of radioactive material or a dye. Furthermore, various chemical tests, such as determination of glucose, protein, and electrolytes, performed on the secreted material have been advocated for the differentiation between CSF leakage and other secretions (2). The injection methods are risk-bearing for the patient and the above methods yield a considerable chance of false-positive or false-negative results (3). Nowadays O -sialotransferrin, also named β2-transferrin, asialotransferrin, or the τ fraction, is an accepted marker protein for the detection of CSF in excretions from the nose or the ear, or from head or neck wounds. O -Sialotransferrin is not strictly unique to CSF. The protein can also be demonstrated in human aqueous humor (4) and in perilymph fluid (5)(6)(7). It is also found in the blood of patients with chronic liver diseases (8)(9), with an inborn error of glycoprotein metabolism (10), and with a genetic variant of transferrin (8)(11)(12). The protein can be detected by direct immunofixation of transferrins after agarose electrophoresis (high-resolution electrophoresis, HRE) (3)(13) or after blotting (1)(14)(15). The method we used up to now was the agarose/immunofixation method according to Zaret et al. (3). We used the Paragon High Resolution Electrophoresis kit (HRE kit, Beckman Instruments) and an anti-transferrin reagent (Beckman Array® System, Beckman Instruments). One disadvantage of this fast method is the high …

Background: Laboratory testing in suspected α-1-antitrypsin (A1AT) deficiency involves analysis of A1AT concentrations and identification of specific alleles by genotyping or phenotyping. The purpose of this study was to define and evaluate a strategy that provides reliable laboratory evaluation of A1AT deficiency.Methods: Samples from 512 individuals referred for A1AT phenotype analysis were analyzed by quantification, phenotype, and genotype. A1AT concentrations were measured by nephelometry. Phenotype analysis was performed by isoelectric focusing electrophoresis. The genotype assay detected the S and Z deficiency alleles by a melting curve analysis.Results: Of the 512 samples analyzed, 2% of the phenotype and genotype results were discordant. Among these 10 discordant results, 7 were attributed to phenotyping errors. On the basis of these data we formulated an algorithm, according to which we analyzed samples by genotyping and quantification assays, with a reflex to phenotyping when the genotype and quantification results were not concordant. Retrospective analyses demonstrated that 4% of samples submitted for genotype and quantitative analysis were reflexed to phenotyping. Of the reflexed samples, phenotyping confirmed the genotype result in 85% of cases. In the remaining 15%, phenotyping provided further information, including identifying rare deficiency alleles and suggesting the presence of a null allele, and allowed for a more definitive interpretation of the genotype result.Conclusions: The combination of genotyping and quantification, with a reflex to phenotyping, is the optimal strategy for the laboratory evaluation of A1AT deficiency.

Hemoglobin (Hb) H disease is a mild to severe form of α-thalassemia caused by the absence/inactivation of three of four α-globin genes. As a result, there are insufficient α-globin chains to form HbA (α2β2), the excess β-globin chains forming unstable HbH (β4) (1), which precipitates and attaches to the erythrocyte membrane to cause membrane dysfunction and hemolysis (2). Genetically, there are two types of HbH disease, deletional HbH disease caused by compound heterozygosity for a double α-globin gene deletion on one chromosome and a single α-globin gene on the other (−α/− −), and nondeletional HbH disease caused by compound heterozygosity for a double α-globin gene deletion and a point mutation or small deletion/insertion on a third α-globin gene (αTα/− − or ααT/− −) (2). Generally, patients with nondeletional HbH disease present with a more severe phenotype than those with deletional HbH disease (1)(2). HbH disease is particularly prevalent in Southeast Asia because of the high frequency of α-thalassemia carriers in these areas (1)(2). In northern Thailand, ∼1.5% (1 in 65) of babies are expected to be born with HbH disease (3). In addition, nondeletional HbH disease with αConstant Spring (HbHCS) is very common in Thailand, where ∼40–50% of patients with HbH disease have nondeletional HbH with αConstant Spring (4). Since the 1980s, isoelectric focusing (IEF) has been widely used in the identification of hemoglobin variants and in the diagnosis of HbH disease and HbBarts hydrops fetalis because of the good separation of HbBarts, HbH, and HbCS tetramers in IEF gels. The accuracy of IEF in HbH disease classification, however, is unknown. To evaluate the accuracy of IEF in predicting α-globin genotype, we analyzed the genotypes of 110 Thai patients identified as having either deletional …

To the Editor: Carbohydrate-deficient transferrin (CDT)1 is a biomarker of growing importance in the assessment of alcohol abuse after conviction for drunk driving. CDT is a more specific indicator for alcohol than traditional liver function tests and is used for identification and follow-up of chronic high alcohol consumption (1). Various methods have been introduced for assaying CDT in serum, including isoelectric focusing, ion-exchange chromatography, HPLC, capillary zone electrophoresis (CZE), and latex enhanced immunonephelometry (1, 2). Measuring the CDT percentage (%CDT) in a forensic context demands CZE or HPLC methodology, because it provides high-resolution separation of serum transferrin (Tf) isoforms and allows the detection of genetic variants and glycosylation disorders. In some cases, the interpretation of CDT results is hampered by the presence of mutant Tf (3). In addition to wild-type Tf (C), D variants (cathodal to C) and B variants (anodal to C) have been described (1). The allele frequencies of the Tf subtypes vary among populations of different ethnicities (4). Exact measurement of D variants of CDT is difficult, however, because di- and trisialylated Tf may coelute with the tetrasialylated D peak …

Among more than 800 hemoglobin (Hb) variants currently described in the HBVar database of the Globin Gene Server (1), variants with elongated chains are very rare. Standard protein techniques such as ion-exchange HPLC and isoelectric focusing (IEF) on polyacrylamide gel can detect many Hb variants (2), but correct identification of single mutated, inserted, or deleted amino acid residues requires more sophisticated techniques, such as electrospray ionization mass spectrometry (ESI-MS) or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (3)(4)(5). The interpretation of DNA sequencing in the presence of inserted nucleotide sequences in the heterozygous state can be difficult and requires direct and reverse sequencing. Protein analysis by MS can be used to check results from DNA sequencing and also can detect posttranslational changes such as acetylation (NH2 terminus), deamidation, methionine oxidation, Hb addition products, or artifacts. ESI-MS and MALDI-TOF MS combined with specific tryptic digestion for peptide mass mapping are rapid and sensitive techniques to confirm inserted amino acid residues. We applied these techniques to the identification of a novel Hb variant with a five-amino acid insertion in the β-globin chain. These techniques allowed us to confirm the DNA sequencing results. Standard Hb analysis was performed by ion-exchange HPLC, IEF on polyacrylamide gel, and reversed-phase (RP)-HPLC of globin chains (6)(7). RP-HPLC was performed with a Vydac C4 analytical column (The Separations Group) with CH3CN–H2O containing 1 mL/L trifluoroacetic acid (TFA) as the mobile phase (7 …