Hemoglobin Rambam (beta69[E13]Gly-->Asp), a pitfall in the assessment of diabetic control: characterization by electrospray mass spectrometry and HPLC.

Hemoglobin (Hb) Rambam, or beta69[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 beta NH2 terminus occurred at an almost identical rate in both wild-type and mutant beta-chains. However, the introduction of a carboxyl group at beta69 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.

Heterogeneity of hemoglobin A1d: assessment and partial characterization of two new minor hemoglobins, A1d3a and A1d3b, increased in uremic and diabetic patients, respectively.

We have separated and quantified two new minor hemoglobins named HbA1d3a and HbA1d3b. The level of HbA1d3a was significantly higher in uremic than in non-uremic patients (3.00 +/- 0.50% vs. 1.28 +/- 0.26% of total hemoglobin). It correlated well with carbamylated hemoglobin (r = 0.80, n = 81, p < 0.002) and with plasma urea concentration (r = 0.78, n = 81, p < 0.002). These data and the electrospray ionization mass spectrometric analysis provide strong evidence that HbA1d3a is an alpha-chain modified by carbamylation. The HbA1d3b level an diabetic patients was found to be 1.6-fold that in non-diabetic subjects (3.00 +/- 0.49 vs. 1.90 +/- 0.33). This was attributed to HbA1d3 modified by glycation. Indeed HbA1d3b correlated significantly with HbA1c (r = 0.71, p < 0.002) and with serum glucose level (r = 0.62, p < 0.002). These two new minor hemoglobins may serve as complements for the objective assessment of averaged long-term uremia and glycemia in uremic and diabetic patients.

Chromatographic evaluation of minor hemoglobins: clinical significance of hemoglobin A1d, comparison with hemoglobin A1c, and possible interferences.

Using an HPLC procedure, we evaluated > 10 minor hemoglobins (Hbs) in healthy adults, individuals on long-term aspirin therapy, diabetic subjects, and uremic patients. Hb A1c and Hb A1d3 were the most abundant and important minor Hb components, respectively, accounting for 4.10% +/- 0.50% and 3.46% +/- 0.43% of total Hb in 361 healthy subjects. Acetylated Hb A was a potential interferent in the measurement of Hb A1c. The amounts of both Hb A1d3 and Hb A1carb were significantly increased in uremic patients, indicating that these Hb adducts may be carbamylated. There was a significant correlation (r = 0.69, P < 0.0001) between the amounts of Hb A1d3 and plasma urea in uremic patients. Nonuremic subjects were clearly separated from uremic patients with regard to the Hb A1d3 content of their total Hb. Our data suggest that Hb A1d3 is useful for assessment of the uremic state and that the combination of Hb A1c and Hb A1d3 could be well-suited for simultaneous control of carbohydrate and urea metabolism.