Study of a newly developed high-performance liquid chromatography analyser for glycosylated haemoglobin measurements in blood containing haemoglobin variants in the Japanese population.

BACKGROUND: This study examined the new high-performance liquid chromatography analyser HLC-723GX (GX) and investigated its ability to both measure glycosylated haemoglobin (HbA1c) values and determine whether haemoglobin variants could cause interference with these measurements in the Japanese population. METHODS: For the basic GX examination, the within- and between-run precision, linearity of measurements, correlation of HbA1c values with current systems and the interference of chemically modified haemoglobin were determined. GX interference caused by the haemoglobin variant was examined by analysing 39 clinical laboratory samples that contained haemoglobin variants. RESULTS: Good within- and between-run precision were found, with the coefficients of variation at ≤1.0%. A wide range of HbA1c measurement values were confirmed, with the HbA1c values strongly correlated with the results of the currently used HLC-723G8 system. Chemically modified haemoglobins were prepared by adding glucose, sodium cyanate, acetaldehyde or acetylsalicylic acid to normal blood samples. None of these samples had any influence on the HbA1c values determined by GX. GX analysis showed haemoglobin variants that eluted after HbA0 and were similar to HbD, or HbS had HbA1c values that were close to those measured by boronate affinity chromatography and immunoassay. GX found lower HbA1c values in blood that contained HbE or haemoglobin variants, which elute before or at nearly the same time as HbA0. CONCLUSIONS: GX is useful for the analysis of HbA1c samples that contain HbD, HbS, HbC and haemoglobin variants, even though the elution times are similar. However, a countermeasure is needed in order to avoid overlooking other haemoglobin variants in Japan.

Hb Buffalo [alpha89(FG1)His-->Gln (alpha1)], observed solely and in the presence of an Hb S [beta6(A3)Glu-->Val] heterozygosity.

The hemoglobin (Hb) pattern of a 32-year-old Somali male living in The Netherlands, during routine diabetes mellitus monitoring, showed two more peaks in addition to the characteristic heterozygous Hb A/S pattern. A major peak of 15% faster than Hb A, and a minor one of 10.8%, overlapping Hb A2 and the glycated Hb S1c fraction were present. The patient was not anemic or microcytic but had a low haptoglobin level, possibly indicating a slightly elevated red blood cell (RBC) turnover. Hb S was confirmed by a sickle test and at the DNA level. The DNA sequence of the alpha1 gene revealed a C-->G transversion at position 89, changing the local positively charged histidine to a neutral glutamine. This mutant has been previously described in a Yemenite woman and two apparently unrelated Somali males. Our case is the first showing Hb Buffalo in combination with Hb S and a G6PD deficiency, and is again observed in a Somali. No functional abnormalities associated with mutations at this amino acid residue are reported in the literature. Also, in this case no sign of any hematological abnormalities that could not be explained by the Hb S heterozygosity G6PD deficiency was found. The abnormal alpha chain is expressed at the expected rate and without thalassemic effect or instability. The mutated alpha chain seems to associate with a slight preference to the beta(A) (15%) rather than with the beta(S) counterpart. The sum of both Hb A(Buffalo) and Hb S(Buffalo) results in about 19-20% of total Hb. This figure is in agreement with a stable mutant of the alpha1 gene.

Lack of relation of increased malformation rates in infants of diabetic mothers to glycemic control during organogenesis.

To determine how much insulin-dependent diabetes increases a woman's risk of giving birth to a malformed infant and how that risk is influenced by metabolic control, we followed 347 diabetic and 389 control women who enrolled in the study within 21 days of conception (the early-entry group) and 279 diabetic women who entered later (the late-entry group). We detected major malformations in the infants of 4.9 percent of the early-entry diabetic women, 2.1 percent of the controls, and 9.0 percent of the late-entry diabetic women. Malformation rates were significantly higher among offspring of early-entry diabetic women than among those of controls (odds ratio, 2.45; lower one-sided 95 percent confidence limit, 1.12; P = 0.027), and higher among late-entry than among early-entry diabetic women (odds ratio, 1.91; lower one-sided 95 percent confidence limit, 1.07; P = 0.032). Mean blood glucose and glycosylated hemoglobin levels during organogenesis were not significantly higher in women whose infants were malformed. Hypoglycemia (glucose, less than or equal to 50 mg per deciliter [2.8 mmol per liter]) was not significantly more common in the same group. Hyperglycemia and glycosylated hemoglobin were not correlated with malformation. The data suggest that more sensitive measures are needed to identify the teratogenic mechanisms, or that not all malformation can be prevented by good glycemic control. Despite the increased malformation rate among infants of the early-entry diabetic women, as compared with the controls, the more favorable outcome seen in the former group as compared with the late-entry group justifies the attempt to achieve good metabolic control around the time of conception.

Carbamylation of hemoglobin in vivo with chronic sublethal dietary cyanide: implications for hemoglobin S.

Carbamylation of the hemoglobin in sickle cell anemia has been demonstrated to improve the status of this hemoglobinopathy. Using an animal model of West African human patterns of chronic sublethal dietary cyanide ingestion, 12 miniature swine consuming either 0, 0.4, 0.7, or 1.2 mg of cyanide/kg body weight/day were studied for 24 weeks to determine if this dietary regime could produce irreversible carbamylated hemoglobin. Throughout the study, the hematological status of all animals remained similar; however, the levels of carbamylated hemoglobin as measured by nanomoles of valine hydantoin varied proportionally to dietary sublethal cyanide intakes, indicating that these natural dietary levels could effect an important and presumably permanent modification of the hemoglobin's beta chain. Serum thiocyanate levels were also positively correlated with cyanide ingestion loads (r = 0.83, P less than 0.01). The implications of these findings in swine are important for the millions of humans with hemoglobin S who regularly consume similar levels of dietary cyanide and for our assessments of the biochemical and medical status of hemoglobin S under natural conditions.

Glycosylated hemoglobin in Saudi sickle cell patients with glucose-6-phosphate dehydrogenase deficiency.

Glycosylated hemoglobin (Hb) was measured in 42 Saudi sickle cell patients and in 38 sickle cell patients with glucose-6-phosphate dehydrogenase (G-6-PD) deficiency. A statistically significant decrease in the percentages of glycosylated Hb was found in patients with sickle cell anemia when compared with those SS subjects with G-6-PD deficiency. Since glycosylated Hb is considered as an index of the red blood cell's life span, it is suggested that the enzyme defect in these patients is possibly ameliorating the severity of sickle cell anemia.

Improved column-chromatographic isolation of glycosylated hemoglobin S.

We investigated glycosylated hemoglobin S by means of chromatography on Bio-Rex 70. The selected elution conditions were similar to those described by Trivelli et al. (N Engl J Med 284: 353-357, 1971), except for modified ionic strength and accurate temperature control. This enabled us to isolate a minor hemoglobin fraction whose properties, as determined by chromatography, electrophoresis, and two-dimensional maps of its tryptic peptides, were typical of a hemoglobin S tetramer with blocked N-terminal residues of the beta subunits. The colorimetric test indicated this to be a glycosylated hemoglobin. This procedure is notably improved over previous chromatographic techniques for isolating glycosylated hemoglobin S. Moreover, it can be easily used in any laboratories where Trivelli's method is routinely in use.

Polymerization of binary mixtures of hemoglobin S and carbamylated hemoglobin S.

To study the mode of interaction between hemoglobin (Hb) S and carbamylated Hb S, the kinetics of polymerization of various mixtures of deoxy-Hb S and NH2 termini carbamylated Hb S in concentrated phosphate buffer was determined. These mixtures were found to polymerize with a clear demonstration of a delay time as does each hemoglobin in its pure form. Both the delay and the polymerization times were prolonged as the fraction of carbamylated Hb S was increased. Electrophoretic analysis of the polymer fraction showed that the amount of carbamylated Hb S increased linearly in the polymer phase with increases in the fraction of carbamylated Hb S in the starting mixture. The ratio of Hb S to carbamylated Hb S in the polymer phase was slightly higher than that in the initial solution mixture, suggesting that Hb S polymerizes more easily than carbamylated Hb S. To examine the role of hybrid hemoglobin in the binary mixtures of carbamylated Hb S and Hb S, we compared the rate of polymerization under which hybrid formation may be prevented or allowed to take place. It was found that the rate of polymerization for the mixtures of carbamylated Hb S and Hb S mixed in the oxy state did not differ from that mixed in the deoxy state. In addition, polymerization occurred even when the critical concentration of each component in the mixture was lower than that of either pure Hb S or pure carbamylated Hb S. These results suggest that all hemoglobin species can participate in the nucleation and polymerization steps of mixtures of carbamylated Hb S and Hb S.

Proportion of glycosylated hemoglobin in erythrocytes of a person with hemoglobins A, S, and G Philadelphia.

The apparent proportion of column-chromatographically measured glycosylated hemoglobin in erythrocytes from an individual with hemoglobins A, S, and G was only 2.3% because the slow glycosylated variant hemoglobins were retarded in the column. In contrast, the value for glycosylated hemoglobin was 7.8% by a new cellulose acetate electrophoretic method that includes use of dextran sulfate buffer. The erythrocyte metalloprotein, carbonic anhydrase B, was shown to co-migrate with glycosylated hemoglobin by this technique, however. Thus carbonic anhydrase B, HbF, and HbA all have weak attraction for negative charges at acid pH. We believe that carbonic anhydrase B should contribute significantly (at least 10-20% absorbance) to the HbA1 by this electrophoretic method. We conclude that microcolumn chromatography should remain the method of choice for HbA1 determination, that subject-based reference intervals should be used for HbS or HbC heterozygotes, and that electrophoretic HbA1 methods should be reserved for use with both HbS and HbC homozygotes and HbSC disease.

Agar gel electrophoretic determination of glycosylated hemoglobin: effect of variant hemoglobins, hyperlipidemia, and temperature.

We investigated the effect of temperature, variant hemoglobins, and hyperlipidemia on determination of glycosylated hemoglobin by an electrophoretic method (Clin. Chem. 26: 1598-1602, 1980). We found that: (a) temperature variations ranging from 4 to 30 degrees C were without effect on results obtained by electrophoresis; (b) concurrent determination of glycosylated hemoglobin by electrophoresis and column-chromatography in blood specimens from 150 diabetic patients yielded almost identical mean values for both procedures when operations were carried out at 22 degrees C; (c) electrophoretic determination of glycosylated hemoglobin in whole-blood hemolysate was not affected by concentration of triglycerides; and (d) unlike column-chromatographic procedures, which underestimate the percentage of glycosylated hemoglobin in patients with hemoglobin S and C, the electrophoretic method accurately determined the proportion of glycosylated hemoglobin in these hemoglobinopathies. Evidently, electrophoresis on agar gel is an excellent alternative to cation-exchange column-chromatographic methods for glycosylated hemoglobin.