Hemoglobin variant HbG-coushatta (beta-22 Glu --> Ala) found by dissociation of blood glucose from values of HbA1C measured by HPLC.

OBJECTIVE: Unexpectedly low values of HbA1C, measured by HPLC compared with their blood glucose levels were found in three related persons. We investigated whether this discrepancy was due to abnormal hemoglobin. PATIENTS AND METHODS: HbA1C was measured by latex agglutination and ordinary HPLC. For further examination, a hemoglobin specimen from the 82-year-old female case was prepared and analyzed by PolyCAT A chromatography, ESI/MS and MS/MS. The HbA1C levels of the three cases measured by HPLC were lower than those measured by latex agglutination. The elution profiles on HPLC of the three gave an unusual peak between HbA1C and HbA0. RESULTS: PolyCAT A chromatography revealed two additional peaks which were not present in normal hemolysates. These peaks were revealed to correspond to abnormalities of HbA0 and HbA1C. The amounts of HbA0 and HbX were almost the same (1:0.85) and their glycation ratios were almost equal (5.2% and 5.9%). ESI/MS showed that the woman's intact globin contained an abnormal beta(X)-chain in addition to the normal alpha(A)- and beta(A)-chains. The molecular weight of this abnormal beta(X)-chain was 58 Da lower than that of the normal beta(A)-chain. Glutamate at the 22nd amino acid residue of the beta(A)-chain was replaced by alanine in the abnormal beta(X)-chain. This variant of Hb was revealed to be the same as HbG-Coushatta (beta-22 Glu --> Ala) from the library of variant Hb. Family studies showed that the variant was inherited as a dominant trait. CONCLUSION: The dissociation was due to underestimation of HbA1C in the measurement by HPLC which excluded glycated variant Hb.

[A variant hemoglobin found by dissociation of blood glucose from HbA1c on routine physical examination].

Discrepancy between HbA1c measured by routine HPLC and blood glucose was observed in four out of 340 persons on physical examination. We investigated whether this discrepancy was due to abnormal hemoglobin. HbA1c was measured by routine HPLC and latex agglutination. For further examinations, a hemoglobin specimen was analyzed by weak cation-exchange column(PolyCAT A) chromatography, ESI/MS and MS/MS. HbA1c levels measured by HPLC were about half of those measured by latex agglutination. An abnormal peak between HbA1c and HbA0 was detected on HPLC chromatography in three consanguineous persons. The abnormal hemoglobin from one of the related person was also confirmed by PolyCAT A chromatography. The hemoglobin specimen contained almost same amounts of normal HbA0 and abnormal Hb. Analyses of the globin by ESI/MS and MS/MS revealed that glutamate at the 22nd amino acid residue of the beta-chain was replaced by alanine. This variant was the same as HbG-Coushatta[beta 22(B4) Glu-->Ala]. Family studies showed that the variant was inherited as a dominant trait. The dissociation observed in the case was due to a presence of a variant hemoglobin. In the other case HbA1c levels measured by the two methods were similarly low, and no abnormal peak was observed on HPLC chromatography. Similar studies on the hemoglobin of this case did not disclose any abnormalities. The low values of HbA1c of this case were considered in the lowest region of the normal range.