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Technical Briefs |
Asn], with Decreased Oxygen Affinity, Causes Falsely Low Hemoglobin A1c Values by HPLC
1 Department of Medicine IV (Endocrinology, Metabolism, and Clinical Chemistry) and
3 Department of Medicine II (Hematology), University of Tuebingen, Otfried-Mueller-Strasse 10, D-72076 Tuebingen, Germany
2 Department of Pediatrics, Hemoglobin Facility, University of Ulm, Prittwitzstrasse 43, D-89075 Ulm, Germany
aauthor for correspondence: fax 49-7071-295974, e-mail ulrich.friess{at}med.uni-tuebingen.de
The lysine in position 66 of the ß chain of hemoglobin (Hb) is located within the heme-binding pocket and is functionally important for oxygen binding (1). The lysine is exceptionally prone to glycation (2) and to modification by advanced glycation endproducts. In this journal, Iwamoto et al. (3) recently proposed an automated immunologic assay for Hb that is specifically advanced glycation endproduct-modified at Lys66.
Here we report on a novel Hb variant at this position [ß66(E10) Lys
Asn] found in a 73-year-old Caucasian patient with a 9-month history of type 1 diabetes mellitus secondary to recurrent necrotizing pancreatitis and choledocholithiasis. He was referred to us because of insufficient metabolic control: in the previous 4-month period, his fasting blood glucose (FBG) had been in the range of 6.3–13.6 mmol/L and his postprandial blood glucose had been in the range of 8.8–17.0 mmol/L. On admission, his FBG was 13.6 mmol/L. The blood smear results, complete blood count, reticulocyte count, and other markers for chronic anemia or hemolysis were within the appropriate reference intervals. HbF (0.6%) and HbA2 (2.1%) were not increased.
The initial measurement of HbA1c by cation-exchange HPLC (Tosoh HLC723-GHb A1c Version 2.2; Tosoh/Eurogenetics) gave a markedly low HbA1c value of 6.0% compared with 9.0% measured by the Bayer DCA immunologic method (Bayer Diagnostics). The chromatogram (Fig. 1A
) obtained with the routinely used 3-min HPLC program showed an abnormal peak interfering with the HbA1c fraction but no other major peak suspicious for a heterozygous Hb variant. Under optimized insulin treatment, the patient remained moderately hyperglycemic. Despite the patient’s persistent mild hyperglycemia, in subsequent HbA1c determinations under 3 and 6 months of optimized insulin treatment, the HPLC method consistently gave unexpectedly low HbA1c readings of 4.9% compared with the HbA1c results (6.6% and 6.3%) from the Bayer DCA immunologic method. Table 1
summarizes the laboratory results. In all chromatograms obtained from the patient, the abnormal peak eluting at 1.10 min interfered with proper calculation of the HbA1c fraction and led to a comparable error at various concentrations of HbA1c (Table 1
).
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To elucidate the nature of this interference, we analyzed the Hb sample by extended HPLC. Analysis by anion-exchange chromatography using MONO Q® 5/5 resin (Amersham Pharmacia) with a continuous pH gradient from pH 8.5 to 6.5 at a flow rate of 1 mL/min separated a major fraction (42%) of abnormal Hb (Fig. 1B
). Hb electrophoresis (Beckman Paragon) under alkaline conditions (Tris-borate agarose gels run in barbiturate buffers at pH 8.6) showed an abnormal Hb band anodal to HbA, whereas under acidic conditions (malate agarose gels using malate buffers at pH 6.0) this Hb fraction comigrated with HbF (Fig. 1C
). Sequence analysis of isolated, amplified leukocyte DNA was performed by standard procedures (4) with an ABI Prism sequencing reagent set and a gel sequencing system (Sequencer 377; all from Applied Biosystems). A heterozygous mutation at codon 66 in exon 2 of the ß-globin gene produced a base exchange (AAAAAT) leading to an expected amino acid exchange from lysine to asparagine (Fig. 1D
).
This amino acid substitution was confirmed on the protein level by liquid chromatography–mass spectrometry (LC-MS) of tryptic digests. The digests of wild-type Hb and Hb variant [ß66(E10) LysAsn] were separated by reversed-phase HPLC with a TSK-Gel Super ODS column [125 mm x 300 µm (i.d.); 2-µm bead size]. The buffer system consisted of 0.25 mL/L trifluoroacetic acid in H2O (buffer A) and 0.23 mL/L trifluoroacetic acid and 600 mL/L acetonitrile in H2O (buffer B), with a gradient of 5% B at 0–15 min, 80% B at 120 min, and 95% B at 140 min. The flow rate was 5 µL/min. Peaks were analyzed by an esquire3000plus electrospray ionization mass spectrometer (Bruker Daltronik).
In wild-type Hb, tryptic cleavage at Lys(ß66) produced a 1669.9-Da peptide (ß67)VLGAFSDGLAHLDNLK, which was identified by its characteristic 2+ mass (m/z 835.9) at a retention time of 76.2 min (data not shown). In digests from the heterozygous propositus (Fig. 1E
), we found two different peptides: the 1669.9-Da wild-type peptide (ß67)VLGAFSDGLAHLDNLK (Fig. 1E
, lanes a and b) and a 1783.9-Da marker peptide (ß66)NVLGAFSDGLAHLDNL, which was identified by its characteristic 2+ mass (m/z 892.9) at a retention time of 83.6 min (Fig. 1E
, lanes a and c). This 1783.9-Da marker peptide appeared in the Hb variant [ß66 LysAsn] only, but not in wild-type Hb, and was confirmed to be an amino acid substitution by elimination of the tryptic cleavage site at Lys(ß66) and by the characteristic mass increase of 114 Da, which indicated the addition of an asparaginyl residue to this position.
The oxygen affinity was determined according to the method of Wimberley et al. (5) on a ABL725 blood gas analyzer (Radiometer Copenhagen). Oximetric readings were (37 °C) as follows: pH 7.41; PCO2 = 33.2 mmHg; PO2 = 118 mmHg; sO2 = 96.1%; deoxyhemoglobin = 3.8%; methemoglobin = 3.1%; carboxyhemoglobin = 0%. P50, the PO2 for a given blood sample at which the Hb is half-saturated, was calculated by the ABL725 software according to the method of Siggaard-Andersen et al. (6) and was increased to 40.6 mmHg (Table 1
), indicating a markedly decreased O2 affinity of Hb [ß66(E10) LysAsn].
We describe the third Hb variant in position ß66. Amino acid substitutions in this position are of interest because this lysine is invariant in the wild-type Hbs of all known species (7). In the Perutz three-dimensional model of Hb, Lys66 is the 10th residue in the E helix (E10), and its 
NH2 group forms an ionic bond to one propionic acid group of the ß-chain heme molecule in the unliganded T state but not in the liganded R state (1)(7). Our observation of reduced oxygen affinity in Hb [ß66(E10) LysAsn] shows the functional importance of this ß66 lysyl residue for oxygen binding and is in accordance with findings for the two other Hb variants described to date. The first variant, HbI Toulouse [ß66 LysGlu] (1), is an unstable Hb variant that produces clinical signs of mild anemia. In this variant, oxygen affinity at pH 7.15 was reduced with a P50 of 38 mmHg compared with 30 mmHg for the wild-type Hb, and other abnormalities, such as increased autooxidation, were observed (8). In the other variant, HbI Chico [ß66 LysThr], oxygen affinity at pH 7.40 was altered to a P50 of 38 mmHg (reference interval, 27 ± 2 mmHg), which reduced oxygen binding by 
50%, whereas the cooperativity of oxygen binding and the allosteric effects of protons, anions, and organic phosphates were not changed (7)(9). Similar observations have come from genetically engineered Hbs: the exchange of Lys66 by site-directed mutagenesis to Ser-66, Arg-66, or Thr-66 led to 1.3-, 1.5-, and 2.3-fold reductions in oxygen affinity, respectively, but allosteric properties such as the Bohr effect and the effect of inositol hexaphosphate were well preserved (10).
Overall we describe a novel silent Hb variant with decreased oxygen affinity that may cause diagnostic confusion because of divergent HbA1c readings obtained by different methods. Our findings support the use of additional chromatographic HbA1c methods in the screening for Hb variants. In such a case, additional methods for HbA1c, e.g., immunologic assays or the LC-MS reference method (11) proposed by the IFCC, should be used.
References
Glu: a new abnormal haemoglobin with a mutation localized on the E10 porphyrin surrounding zone. Nature 1969;223:190-191.[CrossRef][Medline]
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-(carboxymethyl)lysine at lysine 66 of the ß chain. Clin Chem 2001;47:1249-1255.Pro]: first report of a family history. Ann Hematol 2001;80:365-367.[Medline]
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Thr) and its isolated ß chains. J Biol Chem 1991;266:23033-23040.Thr]: a new variant with decreased oxygen affinity. Hemoglobin 1987;11:453-464.[Medline]
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The following articles in journals at HighWire Press have cited this article:
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  D. B. Sacks Hemoglobin Variants and Hemoglobin A1c Analysis: Problem Solved? Clin. Chem., August 1, 2003; 49(8): 1245 - 1247. [Full Text] [PDF]
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