of Ponceau. S protein staining. (2). To. Department. ofPathology,Medical College of Georgia, Augusta,. GA30912. 'Address correspondence toJ.S.K.,atB! H222B ...
CLIN. CHEM. 29/4, 713-714 (1983)
Proportion of Glycosylated Hemoglobin in Erythrocytes of a Person with Hemoglobins A, S, and G Philadelphia Jonathan
Seth Krause,’ Mary H. Jonah, Suh-Jen C. Yau, and Mary Trlnh
The apparent proportion of column-chromatographically measured glycosylatedhemoglobinin erythrocytesfrom an indMdual with hemoglobins A, S, and G was only 2.3% because the slow glycosylated variant hemoglobins were retardedIn the column. In contrast,the value for glycosylated hemoglobinwas 7.8% by a new celluloseacetate electrophoretic methodthat 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 attractionfor negativechargesat acid pH. We believe that carbonic anhydrase B should contribute significantly(at least 10-20% absorbance) to the HbA, by thiselectrophoreticmethod. We concludethat microcolumn chromatography should remain the method of choice for HbA, determination,that subject-based reference intervals should be used for HbS or HbC heterozygotes, and that electrophoreticHbA, methods should be reserved for use with both HbS and HbC homozygotesand HbSC disease. AddItional Keyphrases: buffer
carbonate
hemoglobin variant dextran sulfate dehydrase hemoglobinopathy
Recently we subjected a blood specimen from a 25-yearold pregnant black woman to cellulose acetate electrophoresin, for hemoglobinopathy screening. The electrophoretic pattern showed three hemoglobins,in an “SAC” pattern. In the past the patient had been shown to have hemoglobinsA, S, and G Philadelphia (a P2) by a reference laboratory. Because her electrophoretic pattern was sonovel we decided to quantitate the glycosylated hemoglobin. The patient was in good health. She denied a family historyof diabetes mellitus. A recent value for 1-h postprandial blood glucose was 5.78 mmol/L; results of several urinalyses, both recent and less recent, had been normal.
MaterIals and Methods Hemolysates were electrophoresed on cellulose acetate and citrate agar (materials from Helena Laboratories, Beaumont, TX 77704). HbS was confirmed by solubility testing (“Sickle Sol”; Dade Division Am. Hosp. Supply Corp., Miami, FL 33152). Hemoglobins were quantitated after elution from cellulose acetate (1). Glycosylated hemoglobins were determined by (a) a column-chromatographic method (Isolab, Akron, OH 44321) and (b) an electrophoreticmethod (Gelman Sciences, Ann Arbor, MI 48106) with use of celluloseacetate,a pH 6.5 buffer containing dextran sulfate, and densitometry of Ponceau S protein staining (2). To
quantitate the contribution of carbonic anhydrase B to HbA1 we concentrated a hemolysate fivefold(B-iS, Amicon Corp., Lexington, MA 02173) and subjected it to cellulose acetate electrophoresis with densitometry (CDS-200; Beckman Instruments, Inc., Clin. Instr. Div., Brea, CA 92621) at
520 nm. Mean cell volume and hemoglobinwere determined with an automated blood-cell counter that is based on particle-sizing principles (Coulter Electronics, Inc., Hialeah, FL 33010). Blood smears were air-dried and Wright stained. Buffered carbonic anhydrase B (carbonate dehydrase, EC
4.2.1.1; Sigma Chemical Co., St. Louis, MO 63178; 0.5 and 5.0 g/L; Figure 1) was applied at the origin on the cellulose acetate strip usedfor electrophoretic determination of glycosylated hemoglobin.The proportion of glycosylatedhemoglobin in five diabetic patients’ samples was determined both by electrophoreticand chromatographic methods. The data were examined statistically. Results The mean cell volume was 83 fL; the hemoglobin concentration was 118 g/L of blood with 1+ microcytosis evident in the blood smear. The solubility test result was positive. The
acetate electropherograin showedan “SAC” patan “AS” pattern. The proportionof fetal hemoglobin was
