Haemoglobin J-Baltimore can be detected by HbA1c electropherogram but with underestimated HbA1c value.

Glycated haemoglobin (HbA(1c)) is considered the gold standard for assessing diabetes compensation and treatment. In addition, fortuitous detection of haemoglobin variants during HbA1c measurement is not rare. Recently, two publications reported different conclusions on accuracy of HbA(1c) value using capillary electrophoresis method in presence of haemoglobin J-Baltimore (HbJ).Here we describe the fortuitous detection of unknown HbJ using capillary electrophoresis for measurement of HbA(1c). A patient followed for gestational diabetes in our laboratory presented unknown haemoglobin on Capillarys 2 Flex Piercing analyser which was identified as HbJ. HbJ is not associated with haematological abnormalities. High Performance Liquid Chromatography methods are known to possibly underestimate HbA(1c) value in the presence of this variant. This variant and its glycated form are clearly distinguished on electropherogram but HbJ was responsible for underestimating the true area of HbA(1c). Capillary electrophoresis is a good method for detecting HbJ but does not seem suitable for evaluation of HbA(1C) value in patients in presence of HbJ variant.

A Mosaic Expression of a Hb J-Amiens (HBB: c.54G > T; p.Lys18Asn) and its Interference with Hb A1c Analysis.

We report the case of a 56-year-old Caucasian woman in whom hemoglobinopathy screening was triggered following an aberrant Hb A1c analysis. Preliminary diagnosis of the hemoglobin (Hb) variant was obtained through cation exchange high performance liquid chromatography (HPLC) and gel electrophoresis. DNA analysis confirmed the presence of Hb J-Amiens [ß17(A14)Lys→Asn; HBB: c.[54G > C or 54G > T)]. However, an unbalanced ratio between wild type and mutant signal after direct sequencing and a lower than expected percentage of this Hb variant led to the suggestion of a mosaic expression. Furthermore, different methods [capillary zone electrophoresis (CZE), cation exchange HPLC and boronate affinity] were tested to study the possible interference of this variant with Hb A1c measurements. These investigations showed a clinically relevant difference between the methods tested. Hb A1c analysis may lead to the discovery of new Hb variants or mosaicism for previously described Hb variants. This may have genetic consequences for the offspring of carriers and brings about the question of partner testing.

Isolated Hb Providence ß82Asn and ß82Asp fractions are more stable than native HbA(0) under oxidative stress conditions.

We have previously shown that hydrogen peroxide (H(2)O(2)) triggers irreversible oxidation of amino acids exclusive to the ß-chains of purified human hemoglobin (HbAo). However, it is not clear, whether α- or ß-subunit Hb variants exhibit different oxidative resistance to H(2)O(2) when compared to their native HbAo. Hb Providence contains two ß-subunit variants with single amino acid mutations at ßLys82→Asp (ßK82D) and at ßLys82→Asn (ßK82N) positions and binds oxygen at lower affinity than wild type HbA. We have separated Hb Providence into its 3 component fractions, and contrasted oxidative reactions of its ß-mutant fractions with HbAo. Relative to HbAo, both ßK82N and ßK82D fractions showed similar autoxidation kinetics and similar initial oxidation reaction rates with H(2)O(2). However, a more profound pattern of changes was seen in HbAo than in the two Providence fractions. The structural changes in HbAo include a collapse of ß-subunits, and α-α dimer formation in the presence of excess H(2)O(2). Mass spectrometric and amino acid analysis revealed that ßCys93 and ßCys112 were oxidized in the HbAo fraction, consistent with oxidative pathways driven by a ferrylHb and its protein radical. These amino acids were oxidized at a lesser extent in ßK82D fraction. While the 3 isolated components of Hb Providence exhibited similar ligand binding and oxidation reaction kinetics, the variant fractions were more effective in consuming H(2)O(2) and safely internalizing radicals through the ferric/ferryl pseudoperoxidase cycle.

Hemoglobin J--as a cause of congenital hemolytic anemia.

Hemoglobin-J is a rare hemoglobin variant known to be clinically silent most of the times, only to be detected accidentally. Herein, the authors report a case of Hemoglobin-J manifesting as unstable hemoglobin detected during evaluation of hemolytic anemia in an 8 month-old-infant. Cation Exchange-High Performance Liquid Chromatography(CE-HPLC) was used to identify this variant after Hb electrophoresis was reported to be normal.

[Evaluation of Variant II analyzer equipped with the new 270-2101 NU kit (Bio-Rad) for HbA 1c assay]. / Evaluation de l'analyseur Variant II équipé de la nouvelle trousse 270-2101 NU (Bio-Rad) pour le dosage de l'HbA 1c.

HbA(1c) represents a key parameter in the follow-up of glycemic balance in diabetic patients. It may be assayed by different methods, among which high-pressure liquid chromatography (HPLC). We have evaluated a new method available on HPLC Variant II analyzer (BioRad) equipped with the new kit 270-2101 NU. Chromatographic separation is improved, allowing a better identification of peaks. Intra- and inter-assay coefficients of variation are respectively lower than 1.1% and 1.8%. Linearity is excellent from 3.2% to more than 18%. The correlation with the previous method (kit 270-2101) is good: y (% HbA(1c) new kit) = 0.944x (% HbA(1c) previous kit) + 0.299, r(2) = 0.995. There is no inter-sample contamination. This method is less sensitive to interferences frequently found in practice (labile glycated hemoglobin, carbamylated haemoglobin) than the previous one. Validation is possible in more circumstances when an abnormal hemoglobin is present (especially in case of hemoglobin D or E). As the control of analytic quality is a major element for validation and clinical use of HbA(1c) results, the characteristics of this new method make it a well-suited tool for daily laboratory practice.

Alpha78(EF7)Asn-->Asp is a posttranslational modification in Hb J-Singapore [alpha78(EF7)Asn-->Asp;alpha79(EF8)Ala-->Gly].

In Hb J-Singapore, the alpha78(EF7)Asn-->Asp and alpha79(EF8)Ala-->Gly substitutions were initially thought to be genetic mutations. Subsequent studies on other similar hemoglobin variants suggested that the Asn-->Asp change was a posttranslational modification. Nevertheless, lack of DNA evidence made it difficult to confirm the deamidation in Hb J-Singapore. We recently encountered a female patient with Hb J-Singapore trait. In this report, we present electrospray mass spectrometry and DNA data to confirm the alpha78(EF7)Asn-->Asp is indeed a posttranslational modification.

Adult and fetal haemoglobin J-Sardegna [alpha50(CE8)His-->Asp]: functional and molecular modelling studies.

Haemoglobin (Hb) J-Sardegna [alpha50(CE8)His-->Asp] is a haemoglobin variant characteristic of subjects from the island of Sardinia. Here we report a study of the functional properties of both fetal and adult Hb J-Sardegna. The results indicate that adult Hb J-Sardegna displays an oxygen affinity that is higher than that of adult Hb only in the presence of 2,3-diphosphoglycerate (2,3-DPG). On the contrary, at 20 degrees C, the oxygen affinity of fetal Hb J-Sardegna is identical to that of normal fetal haemoglobin, both in the presence and in the absence of 2,3-DPG. A significant difference between these two systems (i.e. a higher oxygen affinity of fetal Hb J-Sardegna) shows up very clearly only when temperature is increased to 37 degrees C. Hence in fetal Hb, the main effect of the amino acid substitution is a decrease in the overall enthalpy change of oxygenation. The results outline the role of the alpha(1)-beta(1) interface in assessing the thermodynamics of oxygen binding. The functional properties of both adult and fetal Hb J-Sardegna have been interpreted at the structural level in light of the results obtained by a computational modelling approach performed in comparison with HbA and Hb Aichi, a variant characterized by a different mutation [alpha50(CE8)His-->Arg] at the same position.

A diabetic case with hemoglobin J-Meerut and low HbA1C levels.

A diabetic patient with hemoglobin (Hb) J-Meerut and low HbA1C levels is reported. An automatic glycohemoglobin analyzer used for the determination of HbA1C revealed an abnormal peak of the peripheral blood obtained from a Japanese female with diabetes. She showed a lower HbA1C level (3.7%) than expected from her fasting plasma glucose (172 mg/dl). High performance liquid chromatography and isoelectric focusing indicated that her abnormal hemoglobin was Hb J-Meerut [alpha 120(H3)Ala-->Glu] and it accounted for 28.3% of the total hemoglobin. Abnormal hemoglobinemia should be considered when a major discrepancy between the levels of HbA1C and fasting plasma glucose is observed.

Structure dynamics of the hemoglobin mutants Hb Hôtel Dieu, HbG Philadelphia, HbJ Mexico, Hb St. Mandé and Hb San Diego, studied by nanosecond-laser-flash photolysis.

The kinetics of the change from the carboxy to the deoxy conformation of the mutated hemoglobins mentioned in the title and of normal human adult hemoglobin were determined from measurements of light absorption changes occurring up to 50 microseconds after nanosecond-laser photodissociation of the corresponding CO complexes. The spectral evolution of the mutated hemoglobins was found to be similar in its main features to that of normal hemoglobin. The kinetics could be decomposed into two phases with rates 1.1-1.8 x 10(6) s-1 and 0.17-0.34 x 10(6) s-1 (except Hb St. Mandé which displayed only the faster phase). Study of the mutated subunits of HbJ Mexico (alpha subunit) and Hb Hôtel Dieu (beta subunit) showed that they convert exponentially to the stable deoxy state after photodeligation at the same rates as the corresponding subunits of normal Hb: 1.1 x 10(6) s-1 (alpha) and 0.3 x 10(6) s-1 (beta). The results indicate that there is no direct correlation between the kinetics of spectral relaxation in the time range studied and the oxygenation properties for these hemoglobins. However, there is some indication that the kinetics are dependent upon the region of mutation.

A new unstable and low oxygen affinity hemoglobin variant: Hb J-Auckland [beta 25(B7)Gly----Asp].

Hb J-Auckland is a new hemoglobin variant with the amino acid substitution beta 25(B7)Gly----Asp. It is mildly unstable and has a low oxygen affinity. The propositus and a son, both heterozygous for Hb J-Auckland, have marginally low Hb values but no apparent clinical symptoms.

Fluorescence studies on the binding of reduced nicotinamide adenine dinucleotide phosphate to human hemoglobin A and its variant hemoglobin Providence.

The affinity constants for the binding of NADPH to human hemoglobin A were directly determined by fluorescence analysis since nucleotide fluorescence is quenched on binding to the protein. The binding constants 6.1 x 10(5), 5.02 x 10(5) and 1.2 x 10(5) were found for deoxyhemoglobin at pH 6.5, 7.0 and 7.5, respectively. Oxyhemoglobin does not bind NADPH significantly. These results are consistent with those found in oxygen-hemoglobin equilibrium experiments. The human hemoglobin variant, Providence-Asp, which has a marked decrease in 2,3 DPG affinity was also investigated. NADPH does not bind to the variant suggesting that the Lys B 82 residue is of fundamental importance to nucleotide binding and showing that the binding site is the same as that of 2,3 DPG or other organic polyphosphate, allosteric modulators of hemoglobins. Experiments of inositol hexaphosphate (IHP)-NADPH site competition corroborate these results.

Fluorescence studies on the binding of reduced nicotinamide adenine dinucleotide phosphate to human hemoglobin A and its variant hemoglobin providence

The affinity constants for the binding of NADPH to human hemoglobin A were directly determined by fluorescence analyssis since nucleotide fluorescence is quenched on binding to the protein. The binding constants 6.1 x 10**5, 5.02 x 10**5 and 1.2 x 10**5 were found for deosyhemoglobin at pH 6.5, 7.0,respectively. Oxyhemoglobin does not bind NADPH significantly. These results are consistent with those found in oxygen-hemoglobin equilibrium experiments. The human hemoglobin variant, Providence-Asp, which has a marked decrease in 2,3 DPG affinity was also investigated. NADPH does not bind to the variant suggesting that the Lys B 82 residues is of fundamental importance to nucleotide binding and showing that the binding site is the same as that of 2,3 DPG or other organic polyphosphate, aloosteric modulators of hemoglobins. Experiments of inositol hexaphosphate (IHP)-NADPH site competition corroborate these results

[Discovery of hemoglobin J Baltimore in an episode of methemoglobinemia in an infant]. / Découverte d'une hémoglobine J Baltimore au cours d'un épisode de méthémoglobinémie chez un nourrisson.

A case of a heterozygote for Hb J Baltimore is reported in a French family. This variant hemoglobin was coincidentally discovered during an episode of methemoglobinemia in a 6-week-old baby. The father and one of the brothers were also carriers of the trait. Hematological findings for all of them were normal. As Hb J Baltimore is a frequently occurring hemoglobin variant, we discuss: its possible role in the appearance of methemoglobin, and whether this mutation in different racial groups (Caucasians of West Europe, Canadians, and American blacks) has a common origin or more probably arises from a number of independent mutations.

[Familial polycythemia caused by high oxygen affinity hemoglobin. 2nd world case of hemoglobin J Providence]. / Polyglobulie familiale par hémoglobine hyperaffine pour l'oxygène. Deuxième cas mondial d'hémoglobine J Providence.

The cas observed in Auvergne and reported here raises the aetiological problem of polycythaemias. Young subjects with polycythaemia should be investigated for congenital anomaly of oxygen transport by measuring P50 and 2,3-DPG, which provides information on the oxygen-carrying capacity of haemoglobin. When confronted with familial polycythaemia due to high oxygen affinity haemoglobin, clinicians must know that a cause-effect relationship is not always demonstrable since other factors, such as tobacco-smoking in this particular case, may intervene.