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Clinical Chemistry 44: 2172-2177, 1998;
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(Clinical Chemistry. 1998;44:2172-2177.)
© 1998 American Association for Clinical Chemistry, Inc.

Endocrinology and Metabolism

Hemoglobin Rambam (ß69[E13]Gly->Asp), a pitfall in the assessment of diabetic control: characterization by electrospray mass spectrometry and HPLC

E. Bissé1,a, N. Zorn2, A. Eigel3, M. Lizama1, P. Huaman-Guillen1, W. März1, A. Van Dorsselaer2, and H. Wieland1

1 Department of Clinical Chemistry, University Hospital, Hugstetterstrasse 55, D-79106 Freiburg, Germany.

2 Laboratoire de Spectrometrie de Masse Bio-Organique URA31, CNRS-Université Louis Pasteur, Faculté de Chimie, F-67008 Strasbourg, France.

3 Institut für Humangenetik Westfälische Wilhelms-Universität, D-48149 Münster, Germany.
a Author for correspondence. Fax 49-761-270-3444.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hemoglobin (Hb) Rambam, or ß69[E13]Gly->Asp, has been identified in a German woman also suffering from non-insulin-dependent diabetes mellitus and chronic obstructive pulmonary disease. This is the first observation of this Hb variant in a German family thus far. The detailed evaluation of its structure using electrospray mass spectrometry revealed new minor glycohemoglobin components and showed that the attachment of glucose to the ß NH2 terminus occurred at an almost identical rate in both wild-type and mutant ß-chains. However, the introduction of a carboxyl group at ß69 seems to increase the glycation of {epsilon}-amino groups of lysine residues. The glycemic state in the propositus was well reflected by the total glycohemoglobin concentrations but not by the Hb A1C values, which did not reflect hemoglobin glycation in this patient. This case demonstrates that Hb A1C cannot be used reliably in the management of diabetic patients carrying Hb variants such as Hb Rambam. Functional studies of the whole blood of the heterozygous carrier demonstrated extremely low oxygen affinity, which may have been caused by increased 2,3-diphosphoglycerate related to chronic obstructive pulmonary disease and hyperthyroidism. None of the clinical symptoms could be directly associated to Hb Rambam.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Hemoglobin (Hb)1 Rambam or J-Cambridge was first reported more than three decades ago in a Beduin family (1) from Israel and in an English family (2). In these reports, preliminary clinical, hematologic, and electrophoretic data were provided. No subsequent reports of this Hb variant have been published, nor have the quantity of the mutant in heterozygous carriers or the functional properties of Hb Rambam been illustrated more fully during ensuing years (3).

In this study, electrospray ionization mass spectrometry (ESMS) enhanced by reversed-phase HPLC (rp-HPLC) was used to further characterize this rare Hb variant found in a German woman and her daughter. These detailed structural and functional studies complement the original report. In addition, the influence of this variant on the monitoring of diabetes mellitus using glycohemoglobin (GHb) isoforms is documented.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
blood samples
Samples collected in Vacutainer Tubes with EDTA as anticoagulant were obtained from the propositus and her daughter.

Hematologic and biochemical characteristics were determined by standard methods. Hb instability was measured using heat stability and isopropanol stability tests (4). Red cell hemolysates were studied by isoelectric focusing performed on agarose gel. The amounts of Hb A, Hb F, Hb Rambam, and Hb A2 were quantified by cation-exchange HPLC (5). The quantities of the globin chains were determined by rp-HPLC (6). The oxygen-binding properties of whole blood were studied as reported previously (6). The 2,3-diphosphoglycerate (DPG) concentrations were determined enzymatically in whole blood with commercially available kits (Sigma Chemical Co.).

dna analysis
Genomic DNA was isolated from white blood cells using disposable columns from QIAGEN. PCR was used to amplify two fragments of the ß-globin gene, the first fragment extending from position 161 5' to the transcription initiation site to position 113 in intron 2 (773 bp), the second fragment amplifying from position 596 in intron 2 to position 83 3' to the polyadenylation signal (565 bp). After purification of the fragments on agarose gel, single-stranded DNA was obtained by asymmetric amplification with one primer in excess (7). Sequencing of the amplified single-stranded DNA was performed using T7 DNA polymerase, [a-S]-dCTP (Pharmacia), and the sequencing primer SR15(5'-ACAGGTTTAAGGAGACCAATAGA-3') as described previously (8).

esms
ESMS analysis was carried out on a VG BioQ mass spectrometer (Micromass) fitted with an electrospray source operating at atmospheric pressure and succeeded by a triple quadrupole analyzer. The solvent was a mixture of 500 mL/L water–500 mL/L acetonitrile containing 10 mL/L formic acid. The sample diluted with the solvent was introduced into the ionization chamber through a silica capillary of 75-µm internal diameter at a flow rate of 6 µL/min. The potential applied between the end of the capillary and the first electrode was 3.2 kV. The solvent was evaporated using a flow of nitrogen heated at 70 °C. The calibration was performed using horse heart myoglobin [2 µmol/L (2 pmol/µL)], and the scanning ranged from m/z = 250 to m/z = 1800 Da. For the liquid chromatography–mass spectrometry experiments, the eluate from the column was split so that 10 µL/min was introduced in the ESMS and the remaining 190 µL was collected after ultraviolet detection at 214 nm, as described elsewhere (9). Data processing was performed using standard software provided by the manufacturer.

Hemolysates were prepared as reported previously (5). Isolation of a sufficient quantity of ßRambam required a semipreparative rp-HPLC with a 200 x 9 mm, 5 µm, C4 Multospher column (Ziemer). Minor Hbs (Hb X1 and Hb A1) were isolated by cation-exchange HPLC using a 200 x 9.4 mm, 5 µm, PolyCAT A column. Nonglycated Hb and GHb were isolated by affinity chromatography on boronate gel (10). For structural studies, the isolated fractions were concentrated using membrane filtration.

The modified ß-chain was digested using endopeptidase Lys-C (Boehringer Mannheim), which cuts selectively in the NH2 terminus of the lysines. One hundred micrograms of the purified ß-chain was dissolved in 40 µL of buffer (0.1 mol/L Tris-HCl, 0.02 mol/L CaCl2, pH 8.18) and 10 µL of acetonitrile. Lys-C endopeptidase was then added to the solution at an enzyme to protein ratio of 1:20, and the mixture was incubated for 5 h at 37 °C. The peptides thus obtained were analyzed by liquid chromatography–mass spectrometry. The different peptides were separated by rp-HPLC on a Nucleosil 300, 5 µm, C18 column using an acetonitrile gradient (0–60% acetonitrile at a gradient of 1%/min) in a mixture of water containing 1 g/L trifluoroacetic acid and acetonitrile containing 800 mg/L trifluoroacetic acid. Mass measurement allowed identification of all peptides, which were collected. The mutant peptide was sequenced using Edman degradation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
the patient
The index case was a 77-year-old diabetic German woman whose Hb variant was detected during the assessment of her glycemic control. She suffered from chronic obstructive pulmonary disease, hyperthyroidism, hypercholesterolemia, and coxarthritis. She had undergone thyroidectomy and revealed a pronounced degeneration of the dorsal spine with marked postural abnormality. Her blood glucose and fructosamine were 9.80 mmol/L and 350 µmol/L, respectively; however, her Hb A1C was extremely low, 3.5% as measured by Tina-quant, an Hb A1C immunoassay (Boehringer Mannheim). The concentration of her total GHb determined by affinity chromatography was 10.86%. Her ferritin (488 mg/L) and {gamma}-glutamyltransferase (42 U/L) were increased. Hematologic data indicated no abnormalities (not shown). The mutant Hb was separated by cation-exchange chromatography on PolyCAT A. The same Hb variant was found in her 55-year-old daughter. The daughter's medical history included hypertension, hyperthyroidism, and a moderate hypercholesterolemia. She also had a degeneration of the dorsal spine associated with marked postural abnormality.

Hb ANALYSIS
Isoelectric focusing revealed an Hb variant moving anodic to Hb A (not shown). The analysis carried out with cation-exchange HPLC using a PolyCAT A column showed two Hb variant fractions, Hb X1 or Hb R1C and Hb X0 (Fig. 1 top). The concentrations of these Hb variants were 4.38% and 42.50%, respectively. Globin chain analysis by rp-HPLC showed a mutant globin (ßX) eluting behind the wild-type ßA-chain, thus elucidating increased hydrophobicity (not shown). The relative quantity of ßX in the heterozygote was 51.55% of the total ß-chains (ßX ßA). The separation of the GHbs by boronate affinity chromatography, followed by cation-exchange HPLC (Fig. 1 bottom), showed five GHb fractions corresponding to Hb X, Hb A, and Hb A2, respectively. The proportions of these GHbs are shown in Fig. 1 (bottom).



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  		Figure 1. Separation by cation-exchange HPLC of the whole red cell hemolysate from the propositus (top) and the GHb fraction obtained by affinity chromatography (bottom).

oxygen affinity and stability studies
There was a rightward shift of the oxygen dissociation curve of whole blood with a P50 of 66 mmHg. This indicates an extremely low oxygen affinity compared with healthy controls (P50, 25–29 mmHg). Red cell 2,3-DPG was 12.1 mmol/L (reference range, 4.5–6.2 mmol/L).

esms analysis
The hemolysate from the propositus was analyzed, and the resulting profile (Fig. 2 ) revealed three major peaks. The largest peak, with a mass of 15126 ± 0 Da corresponded to an unmodified {alpha}-chain (expected mass, 15126.4 Da). The two other major peaks (at 15867 ± 1 and 15925 ± 1 Da), with close intensities, can be attributed to an unmodified (expected mass, 15867.3 Da) and a modified ß-chain with a mass excess of 58 Da. In view of the isoelectric focusing mobility of the variant, which showed an increase of charge, the rise of the mass could have resulted either from a Gly->Asp or an Ala->Glu replacement. To identify which mutation was responsible for the modified ß-chain and to determine the exact position of this mutation, the modified ß-chain was purified by chromatography and submitted to further analysis. It was subjected to cleavage with endopeptidase Lys-C, and the resulting peptide mixture was analyzed by liquid chromatography–mass spectrometry. All chromatographic peaks detected exhibited masses corresponding to Lys-C digest peptides expected from the sequence of the unmodified ß-chain. However, the peptide L7 (VLGAFSDGLAHLDNLK) with an expected mass of 1670 Da was missing, and a peptide with a mass of 1728 Da was present. This peptide was collected and submitted to Edman degradation, which yielded a sequence corresponding to peptide L7 with an asparagyl residue in place of a glycyl residue at position 3. This clearly indicated an exchange of glycine by aspartic acid at position 69 of the ß-chain.



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  		Figure 2. Electrospray mass spectrum of the hemolysate of the proband.

The multiply charged ion spectrum was converted to a real mass scale spectrum. The major peak at 15126 Da corresponds to the wild-type {alpha}-chain (expected mass, 15126.4 Da); two other peaks (at 15867 and 15925 Da) with close intensities correspond to the wild-type (expected mass, 15867.3 Da) and mutant ß-chains, respectively.

GHb fractions separated by cation-exchange HPLC and by affinity chromatography were collected and analyzed by ESMS. Fig. 3 illustrates the glycation of both unmodified and modified ß-chains. Fig. 3 , A and B, shows peaks of the glycated ß-chains from isolated Hb X1 (16089 Da) and Hb A1C (16027 Da), respectively. Fig. 3C elucidates the distribution of the glycoforms in the GHb fraction. Peaks from {alpha}-chains (15127 Da), both unmodified (15867 Da) and modified (15924 Da) ß-chains, and their glycoforms (15288 Da, 16029 Da, and 16087 Da, respectively) were detected. The percentage of glycation of the modified ß-chain was higher than that of the unmodified ß-chain (33.18% vs 21.67% of the total GHbs, respectively). This result is consistent with the data obtained by cation-exchange HPLC (Fig. 1Up , bottom). Expected and measured molecular masses for the unmodified and modified globin chains are listed in Table 1 .



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  		Figure 3. Distribution of glycoforms in isolated Hb fractions (for interpretation see Table 1Up ).

(A) Mass spectrum of the minor Hb A1C isolated by cation-exchange HPLC. (B) Mass spectrum of the minor Hb Rambam (HbX1) isolated by cation-exchange HPLC. (C) Electrospray mass spectrum of the GHb fraction.


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  		Table 1. Expected and measured molecular masses (Da) for the free and modified globin chains shown in Fig. 3Up .

dna analysis
Direct sequencing of the amplified DNA from the propositus confirmed the nucleotide change from GGT to GAT at codon 69 of the ß-gene, which corresponds to a Gly->Asp replacement at position 69. The analysis of the sample from the daughter gave the same results.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The use of mass spectrometry and DNA analysis enabled us to confirm the mutation and to detect the variant sites. This is thus far the first observation of Hb Rambam in a German family. The combination of PolyCAT A HPLC and boronate affinity chromatography along with ESMS analysis allowed us to determine the distribution of ketoamine-linked glucose at amino groups of the {alpha} and ß globin chains. Our data indicate that the attachment of glucose to the ß NH2 terminus was approximately the same for both wild-type (Hb A1C) and mutant (Hb X1) Hbs. On the other hand, there was a substantial difference in GHb values between Hb X0 and Hb A0. The high GHb percentage of Hb X0 suggests that the replacement of a glycine residue at E69 by aspartate may induce a decrease of the pKa on the carboxyl side of some lysine residues of the ß-chain (e.g., ß66 Lys-Lys), which thereby become more reactive (11).

Hb A1C is widely accepted as the most useful tool for monitoring the glycemic state in diabetic patients. In addition, overestimation or underestimation of this marker may occur in samples containing mutant Hbs, because several HPLC methods lack the resolution necessary to differentiate mutant Hb components (12). However, the measurement of glycated mutant Hbs in addition to Hb A1C in diabetic patients with congenitally modified Hb has not received wide attention (13), particularly since the introduction of the Hb A1C immunoassays (14). Our data illustrate that in the presence of Hb Rambam, the determination of Hb A1C alone is not appropriate for monitoring glycemic control. Indeed, in our patient Hb A1C values determined by ion-exchange HPLC as well as by immunological methods did not reflect the glycemic state, which nevertheless was reflected by the sum of the minor Hbs (Hb X1 Hb A1C) and by the total GHbs. This case points out the necessity of measuring GHbs by affinity chromatography or by high-resolution HPLC in patients carrying mutant Hb variants. Moreover, Hb glycation is not suitable for monitoring glycemic control in carriers of Hb variants with an acetylated NH2 terminus, such as Hb Raleigh, Hb Long Island, and Hb A2 Niigata (15).

Of the 12 known Gly->Asp substitutions in the ß-chains (3), 2 Hb variants, Hb Moscva (16) and Hb J-Auckland (17), have been reported as exhibiting molecular instability with decreased oxygen affinity accompanied by mild anemia. In these two variants, the aspartate residue introduced at ß24(B6) and ß25(B7), respectively, seems to cause a substantial change in the three-dimensional arrangement of the affected subunits. The results reported earlier for Hb Rambam (1) and our data show that this variant is stable and not associated with anemia. It would seem likely that the aspartyl residue in position 69 is not involved in any contact between chains or with the heme group and thereby exerts no molecular disturbance. Hence, no explanation for the extremely low oxygen affinity of whole blood observed in the propositus is evident from the hematological and structural data. 2,3-DPG is an integral part of the molecular mechanism of oxygenation of Hb. Therefore, its concentration in the index patient and her daughter is sufficient to account for the observed decrease in oxygen affinity of whole blood. This increase of the 2,3-DPG concentration may be related to the chronic obstructive pulmonary disease and hyperthyroidism diagnosed in our patients. This is in agreement with data reported earlier in patients with similar clinical states (18). From the clinical point of view, the increased 2,3-DPG effect on a patient's Hb will probably enhance the response to certain physiological or pathological situations. Considering the complex and clinically heterogeneous conditions in our patients, it is difficult to demonstrate whether the resulting decrease in oxygen affinity is a major advantage. There is no clear evidence connecting the clinical features in our patients with Hb Rambam, and it is particularly doubtful that the structural alteration of Hb contributed to diabetes mellitus (19). In addition, marked hypercholesterolemia was also reported in several members of the family with Hb Malmö, which is a high-affinity Hb variant (20). Therefore, the analogy of the clinical pattern of the propositus and her daughter could be explained by other genetic linkages rather than by the presence of Hb Rambam.


   Footnotes
 
1 Nonstandard abbreviations: Hb, hemoglobin; ESMS, electrospray ionization mass spectrometry; rp, reversed-phase; DPG, diphosphoglycerate; and GHb, glycohemoglobin.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Salomon H, Tatarski I, Dance N, Huehns ER, Shooter EM. A new hemoglobin variant found in a Beduin tribe: Hemoglobin "Rambam". Israel J Med Sci 1965;1:836-840. [ISI][Medline] [Order article via Infotrieve]
  2. Sick K, Beale D, Irvine D, Lehmann H. Haemoglobin Gcopenhagen, Haemoglobin Jcambridge two new ß-chain variants of Haemoglobin A. Biochim Biophys Acta 1967;140:231-242. [Medline] [Order article via Infotrieve]
  3. Huisman THJ, Carver MFH, Efremov GD. A syllabus of human hemoglobin variants 1996:198-199 The Sickle Cell Anemia Foundation Augusta, GA. .
  4. Huisman THJ, Jonxis JHP. The hemoglobinopathies: techniques of identification. Clinical and biochemical analysis, Vol. 6 1977:164-167 Marcel Dekker New York. .
  5. Bissé E, Wieland H. High-performance liquid chromatographic separation of human hemoglobin. Simultaneous quantitation of foetal and glycated haemoglobins. J Chromatogr 1988;434:95-110. [ISI][Medline] [Order article via Infotrieve]
  6. Bissé E, Wieland H, Ritschel H. A case of the Hb Regina (ß96(FG3)Leu->Val) in a German male associated with high oxygen affinity and erythrocytosis. Acta Hematol 1991;85:212-216.
  7. Gyllesten UB, Elrich HA. Generation of single stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQa locus. Proc Natl Acad Sci U S A 1988;85:7652-7656. [Abstract/Free Full Text]
  8. Sanger F, Micken S, Coulson AR. DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci U S A 1977;74:5463-5467. [Abstract/Free Full Text]
  9. Van Dorsselaer A, Green B, Jarvis S, Lepage P, Bischoff R, Kolbe HVJ, Roitsch C. Application of electrospray mass spectrometry to the characterization of recombinant proteins up to 44 kDa. Biomed Environ Mass Spectrom 1990;19:692-704. [Medline] [Order article via Infotrieve]
  10. Bissé E, Wieland H. Coupling of m-aminophenylboronic acid to s-triazine-activated Sephacryl: use in the affinity chromatography of glycated hemoglobins. J Chromatogr 1992;575:223-228. [ISI][Medline] [Order article via Infotrieve]
  11. Bunn HF, Forget BG. Hemoglobin: molecular, genetic and clinical aspects 1986:75-82 WB Saunders Philadelphia, PA. .
  12. Weykamp CW, Penders TJ, Muskiet FAJ, van der Slik W. Influence of hemoglobin variants and derivatives on glycohemoglobin determinations, as investigated by 102 laboratories using 16 methods. Clin Chem 1993;39:1717-1722. [Abstract]
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