Preparation of Erythrocyte Membrane

The effect of blood lead on erythrocyte membrane proteins was studied in 28 workers from a scrap lead refining factory and in 18 controls working in railway construction. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the polypeptides in the erythrocyte membrane showed that bands 3 and 4-1 had significantly decreased while bands 2.3, 6, and 7 had significantly increased in the lead workers compared with the controls. For the lead workers, the correlation coefficients between blood lead and bands 2-3 and 3 were r = 0-545 (p < 0.01) and r = -0-51 (p < 0.01) respectively. These results suggest that the decrease in erythrocyte membrane permeability results from a decrease in the membrane transfer protein responsible for band 3. Several studies have been conducted on the effects of lead on erythrocyte membrane. Thus Barltrop and Smith' reported that lead combined with the ,8-chain component of haemoglobin, Mortensen and Kellog2 showed that lead combined with the erythrocyte membrane, and Rubino et al3 reported that the lifespan of erythrocytes had been reduced by lead toxicosis. Selhi and White4 have found both erythrocyte membrane damage and membrane protein conformation changes. Ong and Lee5 studied the in vitro distribution of lead in peripheral blood and reported that 14 1% of 203Pb combined with erythrocyte membrane protein. Others suggested that lead attached to membrane proteins is responsible for the decreased lifespan of erythrocytes.67 The effects, however, of lead on the proteins of the erythrocyte membrane have not been clearly shown. In the present work we used electrophoresis to study membrane proteins in lead workers. Materials and methods SUBJECTS Twenty eight male workers exposed to lead in a lead refining factory were studied. Their mean age was 46-8 (range 30-70). Eighteen men employed in railway construction served as controls. Their mean age was 46-0 (range 28-65). Both groups completed Requests for reprints to: Dr K Fukumoto. Received 2 February 1982 Accepted 1 September 1982 220 a health questionnaire and clinical laboratory tests were performed before the experiment; workers with abnormal liver function test results and endocrinological disorders were excluded. Heparinised blood was taken and placed immediately in iced water. PREPARATION OF ERYTHROCYTE MEMBRANE The method of Dodge et al8 was used. The membrane was prepared and stored at below 4°C. ELECTROPHORESIS (SDS-PAGE) The method of Fairbanks et a19 was used. Reagents were of the highest purity available commercially, and purified water was used. Five percent of SDS gel was used. ANALYSIS OF ELECTROPHOROGRAMS A densitometer (Jyoko Co Ltd, Japan) for serum protein electrophoresis was modified and membrane protein fractions were measured at 570 nm. The bands of the densitometric scan were numbered according to Fairbanks et a19 from the cathode side. ASSAY OF ERYTHROCYTE MEMBRANE PROTEIN The method of Lowry et al "0 was used. DETERMINATION OF BLOOD AND URINE LEAD Samples were dried and ashed then measured by dithizon-polarography."I URINE 8-AMINOLAEVULINIC ACID The method of Tomokuni and Ogata'2 was used. group.bmj.com on July 6, 2017 Published by http://oem.bmj.com/ Downloaded from