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. 2000 Nov;53(11):854–857. doi: 10.1136/jcp.53.11.854

Abnormal haemoglobins, Hb Takamatsu and Hb G-Szuhu, detected during the analysis of glycated haemoglobin (HbA1C) by high performance liquid chromatography

Y Moriwaki 1, T Yamamoto 1, Y Shibutani 1, T Harano 1, S Takahashi 1, T Hada 1
PMCID: PMC1731111  PMID: 11127269

Abstract

Background—During medical checkups of two unrelated female outpatients during their annual health examination and one male inpatient suffering from cardiac failure the glycated haemoglobin (HbA1C) concentrations measured by high performance liquid chromatography (HPLC) were low, in spite of normal fasting plasma glucose concentrations. However, HbA1C concentrations measured by latex immunoagglutination and fructosamine concentrations were within the normal range. Method—Investigations were performed to elucidate the reasons for these discrepancies.

Results—Abnormal haemoglobins, Hb Takamatsu and Hb G-Szuhu, were found. The HPLC chromatogram showed an additional peak near HbA1a+b, which resulted in falsely low HbA1C concentrations. Isoelectric focusing analysis of the patients' haemoglobin disclosed abnormal haemoglobins, which migrated faster than normal HbA1 in the two female patients and slower in the male patient. The cDNA sequence and amino acid analyses of the haemoglobin α-chains and ß-chains indicated the presence of the haemoglobin variant ß 120 Lys→Gln in the two female patients and ß 80 Asn→Lys in the male patient; that is, Hb Takamatsu and Hb G-Szuhu.

Conclusions—These cases show how these silent haemoglobin variants can result in falsely low HbA1C concentration readings when using HPLC.

Key Words: abnormal haemoglobin • high performance liquid chromatography • glycated haemoglobin

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Figure 1 High performance liquid chromatography (HPLC) chromatogram of erythrocyte haemolysates of Hb Takamatsu (patient KM). An additional peak near HbA1a+b caused an underestimation of HbA1C by this method.

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Figure 2 High performance liquid chromatography (HPLC) chromatogram of erythrocyte haemolysates of Hb G-Szuhu (patient ES). An additional peak near HbA1a+b caused an underestimation of HbA1C by this method.

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Figure 3 Isoelectric focusing gel electrophoresis of a control subject, cord blood, and the patient with Hb Takamatsu. Lane 1, electrophoretic pattern of control subject; lane 2, electrophoretic pattern of Hb Takamatsu; lane 3, electrophoretic pattern of cord blood. Abnormal haemoglobin migrating faster than HbA1 is indicated by HbX.

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Figure 4 Isoelectric focusing gel electrophoresis of a control subject and the patient with Hb G-Szuhu. Abnormal haemoglobin migrating slower than HbA1 is indicated by HbX.

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Figure 5 Mutation of amino acid sequence of the Hbß gene at position 120 obtained by means of an ABI PRISM sequencer. The arrow indicates the first nucleotide of the codons for amino acid 120.

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Figure 6 Elution profile of the tryptic digests of aminoethyl abnormal haemoglobin ß of the patient ES (Hb G-Szuhu) by reversed phase high performance liquid chromatography (HPLC).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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