Printer Friendly

Analysis of minor hemoglobins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Major and minor hemoglobin (Hb) [3] components occur in erythrocytes. Examples of major Hb components are HbA, a tetramer consisting of 2 [alpha]- and 2 [beta]-globin chains ([[alpha].sub.2][[beta].sub.2]), which is predominant after childhood and during adult life, or HbF ([[alpha].sub.2][[gamma].sub.2]), which is the main Hb during fetal development. HbF decreases rapidly after birth and becomes a minor Hb. During embryonic development, tetramers of [epsilon]- and [zeta]-chains in combination with [alpha]- and [gamma]-chains are the predominant Hbs. Hb Gower 1 ([[zeta].sub.2][[epsilon].sub.2]), Hb Gower 2 ([[alpha].sub.2][epsilon].sub.2]), and Hb Portland ([[zeta].sub.2][[gamma].sub.2]) serve as oxygen carriers (1). The minor Hb species, designated [HbA.sub.1a] through [HbA.sub.1e] according to their elution order in cation-exchange HPLC (2). consist mainly of [HbA.sub.1], ([[alpha].sub.2][[beta].sup.glyc.sub.2] [HbA.sub.1d3], and [HbA.sub.2] ([[alpha].sub.2][[beta].sub.2]). HPLC analysis of hemolysates of healthy human erythrocytes shows that [HbA.sub.1c] constitutes ~4.4%-5.2% of the total Hb (3). whereas [HbA.sub.1d3] and [HbA.sub.2] account for 3.5% (4) and 2%-3% (5-8), respectively.

The heterogeneity of human Hb arises mainly from posttranslational modifications, principally glycation (9). Glycated Hbs, which are referred to in order of their elution as [HbA.sub.1a1], [HbA.sub.1a2], [HbA.sub.1b1] through [HbA.sub.1b3], and [HbA.sub.1c], result from the nonenzymatic attachment of glucose (in [HbA.sub.1c]), fructose 1,6-diphosphate (in [HbA.sub.1a1]), or glucose 6-phosphate (in [HbA.sub.1a2]) (9,10). Furthermore, Hb can be glycated at multiple sites, including the amino terminus, as well as certain e-amino groups (9). There is no accurate information about the globin chains of [HbA.sub.1d1], [HbA.sub.1d2], and [HbA.sub.1e] available in the literature. Possibly, [HbA.sub.1d1] contains a globin chain modified by acetaldehyde (11). On the other hand, the well-studied [HbA.sub.1d3] is composed of at least 2 components, [HbA.sub.1d3a] and [HbA.sub.1d3b], containing a carbamylated (urea adduct) [alpha]-chain and a glycated minor Hb (4). Additionally, a novel Hb-glutathione adduct, which is increased in patients with diabetes and elutes in the [HbA.sub.1d3] fraction, has been reported (12,13).

Acetylation and Hb adducts of aspirin, vitamin C, penicillin, and acetyl-CoA are additional posttranslational modifications (14). Additionally, Hb adducts with pyruvic acid at the amino terminus of the [beta]-chain (in [HbA.sub.1b]) (15) or glycoinositolphospholipid at the carboxy terminus of the [beta]-chain (16) are known.

The exact quantification of minor Hbs has important diagnostic implications. For example, [HbA.sub.1c] is a marker for diabetes mellitus (1,17); [HbA.sub.1d3a] and [HbA.sub.1d3b] are increased in uremic and diabetic patients, respectively (4); and the controversial influence of Hb-acetaldehyde adducts on [HbA.sub.1d3] in female heavy drinkers has been reported (4,11). HbF is increased in hereditary persistence of fetal Hb, [beta]-thalassemia intermedia, [beta]-thalassemia major, and specific drug treatments (18). Increased HbF is known to inhibit the polymerization of HbS, and the monitoring of HbF concentrations during the follow-up and treatment of patients with sickle cell anemia is mandatory. [HbA.sub.2] is increased in [beta]-thalassemia and in megaloblastic anemia (1) but is decreased in [alpha]-thalassemia, iron deficiency, and sideroblastic anemia (1,18).

Quantification of minor Hbs requires precise and specific methods. A widely applied technique is cation-exchange HPLC, which, however, is known to give falsely increased [HbA.sub.2] values in HbS carriers (5,19-21). In HbD patients, increased (22) as well as decreased (21, 23) [HbA.sub.2] concentrations have been reported. Although incomplete separation of [HbA.sub.2] and HbD explains increased measured [HbA.sub.2] concentrations (22), Suh et al. (19) suggested that coelution of HbS adducts, including glycated HbS, with [HbA.sub.2] also contributes to increased measured [HbA.sub.2] concentrations. Other common Hb analysis techniques include isoelectric focusing, reversed-phase HPLC, and capillary zone electrophoresis (7,14).

Since the development of soft ionization techniques, electrospray ionization and MALDI-TOF MS have provided precise, rapid, and reliable analysis of Hb components. These techniques are well suited to detect globin chain mutations and posttranslational modifications. To date, however, MALDI-TOF MS has seen remarkably little application to Hb analysis (24,25) despite its advantages, such as high sensitivity and short analysis times. Because many of the [beta]-chain variants, including HbC, D-Los Angeles, E, and O-Arab, exhibit mass shifts of only 1 Da, their analyses require high-resolution instruments.

In this study, we used MALDI-TOF MS to investigate globin chains constituting different minor Hbs. We also used this technique to elucidate the minor components in erythrocyte lysates responsible for falsely increased [HbA.sub.2] values in HbS patients.

Materials and Methods


We analyzed a collection of anonymized human blood specimens from a routine chromatography laboratory; therefore, no ethics approval was required. Blood samples had been collected with EDTA or heparin as anticoagulant, stored at 4[degrees]C, and generally analyzed within 24 h. For the determination of [HbA.sub.2] values, we used fresh samples (Hb analysis with PolyCAT A HPLC is a routine analysis of our hospital). In most cases, analysis was performed within 24 h. For the isolation of different Hb fractions, we used some fresh and some previously collected samples (especially the blood samples from patients with rare diseases). [HbA.sub.2] fractions were isolated from fresh samples (in most cases not older than 24 h). We prepared hemolysates for chromatographic analysis by lysing washed erythrocytes in 1 mmol/L KCN.


We performed cation-exchange HPLC with a 200 x 4.6-mm (i.d.) PolyCAT A column (LCC Engineering & Trading GmbH) according to the method of Bisse et al. (26) on a Shimadzu LC-2010C instrument. The HPLC program included (a) a linear gradient from 15% to 65% solvent B in 30 min; (b) a linear gradient from 65% to 100% solvent B in 0.1 min; (c) an isocratic flow of 100% solvent B for 6 min; (d) a linear gradient from 100% to 15% solvent B in 0.1 min; and (e) an isocratic flow of 15% solvent B for 10 min [solvent A, 35 mmol/L Bis-Tris, 1.5 mmol/L KCN, 3 mmol/L N[H.sub.4]C[H.sub.3]COO (pH 6.67); solvent B, 35 mmol/L Bis-Tris, 1.5 mmol/L KCN, 17 mmol/L N[H.sub.4]C[H.sub.3]COO, 150 mmol/L NaC[H.sub.3]COO (pH 7.0)]. We injected 3 [micro]L of sample, monitored peaks at 414 run, and collected fractions of minor Hbs manually. We used WinSTAT[R] software for statistical calculations.


We concentrated the isolated Hb fractions to a final volume of 50 [micro]L with 5-kDa-cutoff Amicon[R] Ultra-4 Centrifugal Filter Devices (Millipore). Before mass spectrometric analysis, we further desalted 10 [micro]L of isolated minor Hb samples with C18 ZipTips[R] (Millipore) and eluted them with 3 [micro]L of 750 mL/L C[H.sub.3]CN in 1 mL/L trifluoroacetic acid (TFA). We used 0.5 [micro]L of the desalted sample for MALDI-TOF MS.


To analyze globin chains, we used the autoflex[R] system (Bruker Daltonics[R]) for MALDI-TOF MS. The mass resolution at 15 kDa was ~750 (m/[DELTA]m). Isolated HPLC fractions were analyzed by the overlayer method. We applied a thin layer of sinapinic acid (saturated solution in ethanol) to a ground-steel MALDI target. We then mixed equal volumes of protein sample and a saturated sinapinic acid solution (in 330 mL/L C[H.sub.3]CN-1 mL/L TFA), applied 0.5 [micro]L of the mixture to the thin layer, and dried the target at room temperature. To analyze the globin digests, we applied 0.5 [micro]L of the sample directly to a 600-[micro]m AnchorChip[TM] (Bruker Daltonics) target and added, on top of the analyze solution, 1.1 [micro]L of a saturated matrix solution consisting of [alpha]-cyano-4-hydroxycinnamic acid (in 330 mL/L C[H.sub.3]CN-1 mL/L TFA), diluted 1:10 in ethanol-acetone (67:33 by volume). After drying the target at room temperature, we performed on-target washing with 2 [micro]L of 1 mL/L TFA.


Globin chains were digested by adding 1 [micro]g of endoproteinase Glu-C (Boehringer Mannheim) or 0.2 [micro]g of trypsin (Promega) and 20 [micro]L of digestion buffer (25 mmol/L N[H.sub.4]HC[O.sub.3], pH 7.8) to the lyophilized sample. The solution was incubated overnight at room temperature (Glu-C digestion) or at 37[degrees]C (trypsin digestion).



A typical PolyCAT A chromatogram of the erythrocyte hemolysate prepared from blood of a healthy donor is shown in Fig. 1A. In addition to the major HbA, a series of minor Hbs are evident, of which the most intense are [HbA.sub.1c]; [HbA.sub.1d3] with its 2 components, [HbA.sub.1d3a] and [HbA.sub.1d3b]; and HbA, Other Hbs, such as [HbA.sub.1a + b]. [HbA.sub.1d1 + 2], and [HbA.sub.1e], have lower intensities and are only partly resolved. Shown in Fig. 1B is a PolyCAT A chromatogram of the lysate from an individual heterozygous for HbS. Typically, an intense peak corresponding to HbS elutes at a retention time of 30 min. MALDI-TOF analysis of the isolated fraction of HbS revealed 2 globin chains, the [alpha]-chain at 15 127 Da and the [[beta].sup.S]-chain at 15 838 Da (Fig. 1B, inset), whereas the typical [alpha]- and [beta]-chains were detected in the HbA fraction (data not shown).


A selection of MALDI-TOF mass spectra of isolated fractions containing separated minor Hbs is shown in Fig. 2. The peaks are assigned according to the expected molecular masses of the globin chains. Table 1 summarizes the globin chains detected in the different fractions. As shown in Fig. 2A, the mass spectrum of a [HbA.sub.1b] fraction isolated from a patient with glycogenose type I includes peaks that correspond to the [alpha]-chain (15 127 Da), the pyruvic acid adduct of the [beta]-chain (15 938 Da), and the latter's decarboxylated form (15 894 Da). The mass spectra in panels B and C of Fig. 2 correspond to the globin chains in the pre-[HbA.sub.1c] and the [HbA.sub.1c] fractions, with glycated [alpha]- and [beta]-chains at molecular masses of 15 289 (Fig. 2B) and 16 030 Da (Fig. 2, B and C), respectively. Analysis of the [HbC.sub.1] fraction isolated from a patient with HbC revealed a very similar mass spectrum for [HbA.sub.1c] (data not shown). [HbC.sub.1], a minor Hb eluting slightly before HbC, contains the characteristic [alpha]-chain and a glycated [[beta].sup.C]-chain that differs by only 1 Da from the typical glycated [beta]-chain, [[beta].sup.glyc].


Shown in Fig. 2D are the globin chains that produce the [HbF.sub.1] peak, a minor Hb in samples with high HbF content. The chains at 15 996 and 16 038 Da are assigned to the [sup.G][gamma]- and the acetylated [sup.G][gamma]-chains, respectively, and both peaks contain a shoulder representing the [sup.A][gamma]- and acetylated [sup.A][gamma]-chains, respectively. Finally, the mass spectra of the partly separated [HbA.sub.1d3a] (Fig. 2E) and [HbA.sub.1a3b] (Fig. 2F) peaks exhibit several different globin chains at 15 127 ([alpha]-chain), 15 868 ([beta]-chain), 15 170, and 15 894 Da. From data reported in the literature, we concluded that the peak at 15 170 Da corresponds to the carbamylated [gamma]-chain (urea adduct), whereas the peak at 15 894 Da is consistent with an acetaldehyde adduct of the [beta]-chain. To confirm these assumptions, we digested the globin chains in the fractions with endoproteinase Glu-C or trypsin. After subsequent MALDI-TOF MS analyses of the peptides, we detected the urea adduct with the N-terminal peptide of the [alpha]-chain (data not shown). The tryptic digest of the peak at 15 894 Da, however, contained no acetaldehyde adduct with the N-terminal peptide of the [beta]-chain. We conclude that a site other than the amino terminus must therefore form an adduct with acetaldehyde.



To investigate the reasons for increased [HbA.sub.2] concentrations in erythrocyte lysates of HbS carriers, we analyzed 32 samples from different heterozygous HbS patients, 42 samples from homozygous HbS patients, and 200 samples from unaffected controls by cation-exchange HPLC (Poly-CAT A). We also analyzed a second control group that included 47 samples from patients with [beta]-thalassemia minor (Table 2). Compared with the control samples, the [HbA.sub.2] concentrations in HbS patients (homozygous and heterozygous) were statistically significantly increased (P <0.0001, Student t-test). In homozygous and heterozygous HbS patients, the concentrations were 4.95% and 4.22% of total Hb, respectively. These concentrations are clearly increased relative to the concentration (2.68%) measured in the control samples. The [HbA.sub.2] concentration in homozygous HbS patients (4.95%) approached that of [beta]-thalassemia patients (5.35%).


We assumed that a minor Hb coeluted with [HbA.sub.2] and thus falsely increased [HbA.sub.2] values in patients with sickle cell anemia. We therefore isolated the [HbA.sub.2] fractions of 12 HbS samples (3 heterozygotes and 9 homozygotes) and 2 [beta]-thalassemia samples. The MALDI-TOF mass spectra of the [HbA.sub.2] fractions with 2 intensive peaks at 15 127 and 15 925 Da correspond to the normal [alpha]- and [delta]-chains, respectively (Fig. 3). Both [beta]-thalassemia samples gave similar spectra. On the other hand, the MALDI-TOF mass spectrum of the [HbA.sub.2] fraction isolated from a patient with HbS included 4 different globin chains with masses at 15 127,15 170,15 838, and 15 925 Da (Fig. 3B). All [HbA.sub.2] fractions isolated from 9 HbS samples gave similar MALDI-TOF mass spectra (data not shown). To exclude artifacts, we purified an isolated [HbA.sub.2] fraction of a heterozygous HbS carrier 3 times by cation-exchange chromatography, and MALDI-TOF analysis revealed the peak at 15 838 Da. As in Fig. 3A, the peaks at 15 127 and 15 925 Da were assigned to the normal [alpha]- and [delta]-chains. The peak at 15 838 Da, moreover, corresponds to the [[beta].sup.S]-chain, whereas the peak at 15 170 Da is consistent with a posttranslationally modified [alpha]-chain. Considering the results obtained for the [HbA.,sub.1d3a] fraction, we assumed that carbamylation of the [alpha]-chain accounted for the mass shift of 43 Da compared with the normal [alpha]-chain. With digestion of the [HbA.sub.2] fraction by endoproteinase Glu-C and subsequent MALDI-TOF MS analysis of the resulting peptides, we confirmed this assumption (data not shown). The 15 170-Da globin chain corresponds to the N-terminal-carbamylated [alpha]-chain.


Traditional methods for Hb analysis, such as chromatography and electrophoresis, reveal only symmetric Hb tetramers because of the rapid dissociation of tetramers to dimers relative to the separation time (27). We confirmed this finding by analyzing a heterozygous HbS sample (Fig. 1B). MALDI-TOF analysis of the isolated HbA and HbS fractions revealed only globin chains of symmetric Hbs, [alpha][beta] (tetrameric structure, [[alpha].sub.2]([[beta].sub.2]) and [alpha][[beta].sup.S] ([[alpha].sub.2]([[beta].sup.S.sub.2]), respectively. We observed no peak for the expected asymmetric tetramer az00S, leading us to conclude that the observed peaks in cation-exchange HPLC all correspond to dimers and not to tetramers. The signals in a PolyCAT A chromatogram therefore do not reflect the real tetrameric Hbs.


Use of MALDI-TOF MS to analyze desalted and concentrated minor Hb fractions allows measurement of the globin chains that build the tetramerc structure of the Hb. We applied our procedure to isolated Hbs detected in Hb samples from healthy individuals and from patients with various disorders that produce hemoglobin variants (Table 1). The mass spectrum of the [HbA.sub.1b] fraction (Fig. 2A) isolated from a patient with glycogenose type I is characterized by 2 posttranslationally modified [beta]-chains, of which the peak with mass 15 938 Da represents the pyruvated [beta]-chain built by nonenzymatic condensation of pyruvic acid with the [beta]-chain amino terminus (1). Additionally, the decarboxylated form of the pyruvated [beta]-chain (15 894 Da) is present. Pyruvate adducts correspond well with increased pyruvate concentrations in patients with glycogen storage disease type I.

The mass spectrum of a pre-[HbA.sub.1c] fraction that elutes slightly before [HbA.sub.1c] (at 30% solvent B), in a sample from a patient with diabetes, is shown in Fig. 2B. In addition to the normal [alpha]-chain, the glycated [alpha]- and [beta]-chains are detectable at 15 289 and 16 030 Da, respectively. We conclude that pre-[HbA.sub.1c] must contain a Hb with at least 1 glycated [alpha]-chain. On the other hand, the [HbA.sub.1c] fraction (Fig. 2C), an important diabetes marker, contains the expected [alpha]-chain and [[beta].sup.glyc]-chain, leading to the known [[alpha].sub.2][[beta].sup.glyc.sub.2] structure. The analysis of a [HbC.sub.1] fraction revealed a mass spectrum very similar to that for a normal [alpha]-chain and a glycated [[beta].sup.C]-chain, which differs by only 1 Da from glycated [beta]-chain (data not shown). In diabetic patients with HbC, therefore, the minor [HbC.sub.1] peak represents a dimer [alpha][[beta].sup.C,glyc] and must be included in the quantification of glycated Hbs.

The mass spectrum of the [HbF.sub.1] fraction, an Hb that appears with high HbF concentrations, exhibits the acetylated [sup.G][gamma]- and [sup.A][gamma]-chains (shoulder of the peak; Fig. 2D). Acetylation as a posttranslational modification occurs frequently in human proteins. Native Hb, however, is not N-terminally acetylated (except for [gamma]-globin) because the N-terminal valine of [alpha]-, [beta]-, and [delta]-globins inhibits acetylation (28). Both nearly identical [gamma]-globin chains are expressed by the [sup.G][gamma]-globin and [sup.A][gamma]-globin genes. In humans at the age of 5 months, [sup.A][gamma]-globin chains become predominant (8). Finally, [HbF.sub.1] and HbF ([[alpha].sub.2][[gamma].sub.2]) are increased in patients with homozygous HbS on hydroxyurea treatment. This drug increases, by as yet unclear processes, the HbF concentration, which inhibits the polymerization of HbS, thereby reducing vaso-occlusive events (29).

[HbA.sub.1d3a] (Fig. 2E) and [HbA.sub.1d3b] (Fig. 2F), which eluted at 45% and 48% solvent B, respectively, could not be separated completely. Of the 4 intense peaks detected in the [HbA.sub.1d3a] fraction, the peak at 15 170 Da corresponds to the carbamylated [alpha]-chain, and the peak at 15 868 Da is consistent with the normal [beta]-chain. We attribute the chains with masses of 15 127 and 15 894 Da to the partly overlapping HbAla3b fraction. [HbA.sub.1d3a] is reported to be significantly higher in uremic than in nonuremic patients (4). These observations correspond well with our findings of a carbamylated [alpha]-chain (adduct of urea to the amino terminus of the [alpha]-chain); on the other hand, the [HbA.sub.1d3b] fraction exhibited 3 major chains, the normal [alpha]- and [beta]-chains and a modified [beta]-chain at 15 894 Da (Fig. 2F). The characteristic [alpha]- and [beta]-chains in this fraction probably arise partly from overlapping of the highly intense HbA peak. [HbA.sub.1d3] is reported to be increased in female heavy drinkers and in alcoholic individuals, but the increase is not statistically significant (11). The first metabolite of ethanol, acetaldehyde, is known to form adducts with Hbs (10). We therefore assume that the peak at mass 15 894 Da could be attributed to the [beta]-chain adduct with acetaldehyde ([[beta].sup.ach]), which corresponds well with the observed mass shift of 26 Da. Occasionally, in patients with diabetes, the glutathionylated [beta]-chain at mass 16173 Da is also detected (data not shown), a finding consistent with other reports (4,12,13) of increased glutathionylated [beta]-chain concentrations that lead to increased [HbA.sub.1d3b] concentrations. Additionally, a minor peak corresponding to a glycated [alpha]-chain (15 289 Da) was detected. Assuming a dimeric structure of PolyCAT A-separated compounds, we conclude that this fraction must contain at least 2 different Hbs, namely [alpha][[beta].sup.ach] and [[alpha].sup.glyc][beta].

The [HbA.sup.1d] fraction is reported to increase slowly with hemolysate age (30). Possibly, carbamylation plays a crucial role in Hb aging. Elucidation of the aging process, however, will require further studies.

HbBarts ([[gamma].sub.4]) and HbH ([[beta].sub.4]), 2 Hbs consisting of only one type of globin chain, are markers for [alpha]-thalassemia. These Hbs elute at 18% solvent B when PolyCAT A HPLC is used. Analysis of the isolated fraction of HbBarts revealed only 1 intense peak at 16 010 Da, corresponding to the [gamma]-chain (data not shown).

We also tried to analyze other fractions containing minor Hbs, such as [HbA.sub.1a], [HbA.sub.1d1 + 2], and [HbA.sub.1e]. Low concentrations and strong overlapping of intense Hbs, however, prevented successful analysis of these components. To identify these minor Hbs, a promising strategy is the analysis of disease samples that exhibit increased minor Hbs as observed in the [HbA.sub.1b] peak isolated from a patient with glycogenose type 1.

We also used analysis of minor Hbs by MALDI-TOF MS to investigate [HbA.sub.2] in HbS carriers. It is well established that [HbA.sub.2] is increased in the presence of the [beta]-chain variant HbS (5,19-21) when analyzed by chromatographic methods. Additionally, [HbA.sub.2] is reported to inhibit polymerization of deoxy-sickle hemoglobin (HbS) in vitro (31). In this study, we analyzed samples from HbS carriers and compared their [HbA.sub.2] values with the values for controls and confirmed [beta]-thalassemia carriers. The [HbA.sub.2] concentrations of the HbS patients were clearly increased compared with those of the controls (Table 2) and agree with those of Shokrani et al. (5). In 1996, Suh et al. (19) undertook a study to investigate the increased [HbA.sub.2] concentrations in greater detail. They assumed that coelution of HbS adducts, including glycated HbS with [HbA.sub.2] on HPLC, was responsible for the increased [HbA.sub.2] value. They were not able, however, to elucidate the supposed coeluting Hb component.

To investigate this proposed HbS adduct, we analyzed isolated [HbA.sub.2] fractions of HbS and [beta]-thalassemia samples with MALDI-TOF MS. In the [HbA.sub.2] fraction of a patient with [beta]-thalassemia (without HbS), the expected [alpha]- and [delta]-chains (Fig. 3A) were present. The different signal intensities of the 2 peaks resulted from distinct mass spectrometric signal responses and not from various amounts of the globin chains. In contrast to the spectrum of the [beta]-thalassemia sample, we found in all investigated HbS samples additional globin chains at 15 170 and 15 838 Da (Fig. 3B). The 15 838-Da globin chain could easily be assigned to the variant [beta]-chain, [[beta].sup.S]. The presence of the [[beta].sup.S]-chain in the [HbA.sub.2] fraction is explained by incomplete separation of an intense HbS peak and the [HbA.sub.2] peak when analyzed with cation-exchange HPLC. In heterozygous HbS samples, we also detected the peak at 15 838 Da, but we could separate the HbS peak and the [HbA.sub.2] peak completely. We therefore conclude that the variant [[beta].sup.S]-chain is participating in coelution of the minor Hb with [HbA.sub.2].

The peak at 15 170 Da must be assigned to a posttranslationally modified [alpha]-chain. Considering the previously found globin chains in [HbA.sub.1d3a]. we suspected that the modification is carbamylation of the [alpha]-chain. Mass spectrometric analysis of the peptides resulting from endoproteinase Glu-C digestion of the [HbA.sub.2] fraction confirmed this assumption (data not shown). From these results, we conclude that the increase in [HbA.sub.2] in patients with HbS cannot be attributed to the presence of glycated [[beta].sup.S], as is commonly accepted, but arises from coelution of the minor Hb [[alpha].sup.carb][[beta].sup.S] with [HbA.sub.2].

We are grateful to Hannes Frischknecht for helpful discussions and important suggestions. We thank Dr. Rowena Crockett for critical reading of the manuscript. We thank Dr. H. Oezahin for providing some of the investigated samples.


(1.) Bunn HE Human hemoglobins: normal and abnormal; methemoglobinemia. In: Nathan DG, Oski FA, eds. Hematology of infancy and childhood, 4th ed. Philadelphia: WB Saunders, 1993:698-731.

(2.) Bisse E, Huaman-Guillen P, Wieland H. Chromatographic evaluation of minor hemoglobins: clinical significance of hemoglobin A1d, comparison with hemoglobin A1c, and possible interferences. Clin Chem 1995;41:658-63.

(3.) Carrera T, Bonamusa L, Almirall L, Navarro JM. Should age and sex be taken into account in the determination of HbA1c reference range? Diabetes Care 1998;21:2193-4.

(4.) Bisse E, Huaman-Guillen P, Horth P, Busse-Grawitz A, Lizama M, Kramer-Guth A, et al. Heterogeneity of hemoglobin A1d: assessment and partial characterization of two new minor hemoglobins, A1d3a and A1d3b, increased in uremic and diabetic patients, respectively. J Chromatogr B Biomed Appl 1996;687:349-56.

(5.) Shokrani M, Terrell F, Turner EA, Aguinaga MD. Chromatographic measurements of hemoglobin A2 in blood samples that contain sickle hemoglobin. Ann Clin Lab Sci 2000;30:191-4.

(6.) Wild BJ, Bain BJ. Detection and quantitation of normal and variant haemoglobins: an analytical review. Ann Clin Biochem 2004;41: 355-69.

(7.) Head CE, Conroy M, Jarvis M, Phelan L, Bain BJ. Some observations on the measurement of haemoglobin A2 and S percentages by high performance liquid chromatography in the presence and absence of a thalassaemia. J Clin Pathol 2004;57:276-80.

(8.) Nagel RL, Steinberg MH. Hemoglobins of the embryo and fetus and minor hemoglobins of adults. In: Steinberg MH, Forget BG, Higgs DR, Nagel RL, eds. Disorders of hemoglobin: genetics, pathophysiology, and clinical management. Cambridge: Cambridge University Press, 2001:197-230.

(9.) Fluckiger R, Mortensen HB. Glycated haemoglobins. J Chromatogr 1988;429:279-92.

(10.) Itala L, Seppa K, Turpeinen U, Sillanaukee P. Separation of hemoglobin acetaldehyde adducts by high-performance liquid chromatography-cation-exchange chromatography. Anal Biochem 1995;224:323-9.

(11.) Hurme L, Seppa K, Rajaniemi H, Sillanaukee P. Chromatographically identified alcohol-induced haemoglobin adducts as markers of alcohol abuse among women. Eur J Clin Invest 1998;28:87-94.

(12.) Al-Abed Y, VanPatten S, Li H, Lawson JA, FitzGerald GA, Manogue KR, et al. Characterization of a novel hemoglobin-glutathiony adduct that is elevated in diabetic patients. Mol Med 2001;7: 619-23.

(13.) Niwa T, Naito C, Mawjood AH, Imai K. Increased glutathionyl hemoglobin in diabetes mellitus and hyperlipidemia demonstrated by liquid chromatography/electrospray ionization-mass spectrometry. Clin Chem 2000;46:82-8.

(14.) Frantzen F. Chromatographic and electrophoretic methods for modified hemoglobins. J Chromatogr B Biomed Sci Appl 1997; 699:269-86.

(15.) Prome D, Blouquit Y, Ponthus C, Prome JC, Rosa J. Structure of the human adult hemoglobin minor fraction Alb by electrospray and secondary ion mass spectrometry. Pyruvic acid as amino-terminal blocking group. J Biol Chem 1991;266:13050-4.

(16.) Niketic V, Tomasevic N, Nikolic M. Covalent glycoinositolphospholipid binding to hemoglobin: a new posttranslational modification of Hb occurring in hyperinsulinism with concomitant hypoglycemia. Biochem Biophys Res Commun 1997;239:435-8.

(17.) Bry L, Chen PC, Sacks DB. Effects of hemoglobin variants and chemically modified derivatives on assays for glycohemoglobin. Clin Chem 2001;47:153-63.

(18.) Cotton F, Lin C, Fontaine B, Gulbis B, Janssens J, Vertongen F. Evaluation of a capillary electrophoresis method for routine determination of hemoglobins A2 and F. Clin Chem 1999;45:237-43.

(19.) Suh DD, Krauss JS, Bures K. Influence of hemoglobin S adducts on hemoglobin A2 quantification by HPLC. Clin Chem 1996;42: 1113-4.

(20.) Whitten WJ, Rucknagel DL. The proportion of Hb A2 is higher in sickle cell trait than in normal homozygotes. Hemoglobin 1981; 5:371-8.

(21.) Dash S. Hb A2 in subjects with Hb D. Clin Chem 1998;44: 2381-2.

(22.) Huisman TH. Combinations of R chain abnormal hemoglobins with each other or with R-thalassemia determinants with known mutations: influence on phenotype. Clin Chem 1997;43:1850-6.

(23.) Cotton F, Gulbis B, Hansen V, Vertongen F. Interference of hemoglobin D in hemoglobin A(2) measurement by cation-exchange HPLC. Clin Chem 1999;45:1317-8.

(24.) Houston CT, Reilly JP. Toward a simple, expedient, and complete analysis of human hemoglobin by MALDI-TOF-MS. Anal Chem 1999;71:3397-404.

(25.) Bajuk A, Michalak L. Some aspects of matrix-assisted laser desorption/ionization analysis of hemoglobin from whole human blood. Rapid Commun Mass Spectrom 2002;16:951-6.

(26.) Bisse E, Wieland H. High-performance liquid chromatographic separation of human haemoglobins. Simultaneous quantitation of foetal and glycated haemoglobins. J Chromatogr 1988;434:95-110.

(27.) Ofori-Acquah SF, Green BN, Davies SC, Nicolaides KH, Serjeant GR, Layton DM. Mass spectral analysis of asymmetric hemoglobin hybrids: demonstration of Hb FS ([alpha]2[gamma][beta]S) in sickle cell disease. Anal Biochem 2001;298:76-82.

(28.) Wada Y. Advanced analytical methods for hemoglobin variants. J Chromatogr B Analyt Technol Biomed Life Sci 2002;781:291-301.

(29.) Bunn HF. Pathogenesis and treatment of sickle cell disease. N Engl J Med 1997;337:762-9.

(30.) Schifreen RS, Hickingbotham JM, Bowers GN Jr. Accuracy, precision, and stability in measurement of hemoglobin A1C by "high-performance" cation-exchange chromatography. Clin Chem 1980; 26:466-72.

(31.) Sen U, Dasgupta J, Choudhury D, Datta P, Chakrabarti A, Chakrabarty SB, et al. Crystal structures of [HbA.sub.2] and HbE and modeling of hemoglobin delta 4: interpretation of the thermal stability and the antisickling effect of [HbA.sub.2] and identification of the ferrocyanide binding site in Hb. Biochemistry 2004;43: 12477-88.


[1] Division of Hematology, and [2] Department of Pediatrics, Division of Clinical Chemistry and Biochemistry, University of Zurich, Zurich, Switzerland.

[3] Nonstandard abbreviations: Hb, hemoglobin; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; and TFA, trifluoroacetic acid.

* Address correspondence to this author at: Department of Pediatrics, Division of Clinical Chemistry and Biochemistry, University of Zurich, Steinwiesstrasse 75, 8032 Zurich, Switzerland. Fax 41-1-266-71-69; e-mail

Received January 7, 2005; accepted March 14, 2005.

Previously published online at DOI: 10.1373/clinchem.2005.047985
Table 1. Major and minor Hbs and the detected globin chains.

 Hb Elution in Globin chains detected by MALDI-TOF
 PolyCAT A MS (mass in Da)
 HPLC, % B

HbH 18 [beta] (15 868)
HbBarts 18 [G.sub.[gamma] (15 996);
 [sup.A][gamma] (16 010)
[HbA.sub.1b] 27 [alpha] (15 127); [[beta].sup.pyr]
[pre-HbA.sub.1c] 30 [alpha] (15 127); [[alpha].sup.glyc]
 (15 289); [[beta].sup.glyc] (16 030)
[HbA.sub.1c] 35 [alpha] (15 127); [[beta].sup.glyc]
 (16 030)
[HbF.sub.1] 30 [alpha] (15 127); [sup.G][gamma]
 (15 996); [sup.A][gamma] (16 010,
 shoulder); acet.
 [sup.G][gamma] (16 038); acet.
 [sup.A][gamma] (16 052, shoulder)
HbF 37 [alpha] (15 127); [sup.G][gamma]
 (15 996); [sup.A][gamma] (16 010)
[HbA.sub.1d3a] 45 [[alpha].sup.carb] (15 170); [alpha]
 (15 127); [beta] (15 868);
 [[beta].sup.ach] (15 894) [beta]-GSH
 (16 173)
[HbA.sub.1d3b] 48 [alpha] (15 127); [[alpha].sup.glyc]
 (15 289); [beta] (15 868);
 [[beta].sup.ach] (15 894)
HbA 52 [alpha] (15 127); [beta] (15 868)
[HbA.sub.2] 60 [alpha] (15 127); [delta] (15 925)
[HbA.sub.2] 60 [alpha] (15 127); [[alpha].sup.carb]
 (15 170); [delta] (15 925);
 [[beta].sup.S] (15 838)
HbS 100 [alpha] (15 127); [[beta].sup.S]
 (15 838)
[HbC.sub.1] 100 [alpha] (15 127); [[beta].sup.c, glyc]
 (16 030) (b)
HbC 100 [alpha] (15 127); [[beta].sup.c]
 (15 868) (b)

 Hb Sample origin

HbH [alpha]-Thalassemia
HbBarts [alpha]-Thalassemia

[HbA.sub.1b] GSD-I (a)
[pre-HbA.sub.1c] Diabetes
[HbA.sub.1c] Diabetes
[HbF.sub.1] Newborn
HbF Newborn
[HbA.sub.1d3a] Control
[HbA.sub.1d3b] Control
HbA Control
[HbA.sub.2] Control
[HbA.sub.2] HbS
[HbC.sub.1] HbC

(a) GSD-I, glycogen storage disease type I; pyr, pyruvic acid
adduct; pyr-C[O.sub.2], decarboxylated pyruvic acid adduct; glyc,
glycated; acet, acetylated; carb, carbamylated; ach, acetaldehyde
adduct; GSH, glutathione.

(b) Because of limited instrument mass resolution, the expected
mass shift of 1 Da, compared with the normal glycated [beta]-chain,
is not detectable with conventional MALDI-TOF instruments.

Table 2. [HbA.sub.2] concentrations in patients with HbS or
[beta]-thalassemia, and in control samples.

 Samples n Mean (range), CV, %
 % of total Hb

Controls 200 2.68 (2.0-3.5) 11
[beta]-Thalassemia minor 47 5.35 (4.1-7.3) 13
HbS heterozygote 32 4.22 (3.6-5.1) 7.6
HbS homozygote 42 4.95 (3.1-7.0) 33
COPYRIGHT 2005 American Association for Clinical Chemistry, Inc.
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2005 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:Hematology
Author:Zurbriggen, Karin; Schmugge, Markus; Schmid, Marlis; Durka, Silke; Kleinert, Peter; Kuster, Thomas;
Publication:Clinical Chemistry
Date:Jun 1, 2005
Previous Article:Multimarker quantitative real-time PCR detection of circulating melanoma cells in peripheral blood: relation to disease stage in melanoma patients.
Next Article:Identification of ghrelin in human saliva: production by the salivary glands and potential role in proliferation of oral keratinocytes.

Terms of use | Privacy policy | Copyright © 2022 Farlex, Inc. | Feedback | For webmasters |