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Erythrocyte membrane fluidity in type 1 diabetes mellitus
Pathology (1983), 15, pp. 49-51
ERYTHROCYTE MEMBRANE FLUIDITY IN TYPE 1 DIABETES MELLITUS MICHAEL A. HILLAND J. M. COURT Department of Dewlopmental Paediatrics, Royal Children's Hospital, Melbourne
Summary Erythrocyte membrane fluidity was determined in a group of type 1 diabetics in varying metabolic control. No difference in membrane fluidity, as measured by fluorescence polarization of 1,6-diphenyI-l,3,5-hexatriene, was found between cells from diabeticsubjects. In addition, nodifference wasdetected in membrane phospholipid and cholesterol content or the ratio of cholesterol to phospholipid in ihe diabetic subjects when compared to controls. The present study suggests that changes in erythrocyte membrane fluidity do not play a major role in the alterations of the physical properties of blood seen in type 1 diabetes mellitus.
Key Mord.v. diabetes mellitus, microvascular disease. erythrocyte membrane fluidity
INTRODUCTION There is increasing speculation that alterations to flow characteristics within the microcirculation are involved in the development of the vascular complications ofdiabetes mellitus. Microvascular flow may be altered by changes in the physical properties of erythrocytes, and such cells from diabetic subjects have been shown to be less deformable' more prone to aggregation3 and more adhesive to endothelial surfaces4 compared to erythrocytes from a non-diabetic population. In type 1 diabetes there may be alterations in plasma concentrations of lipids related to the blood glucose levels achieved in diabetic control.5, The erythrocyte is susceptible to changes in concentration of glucose and lipids in plasma as it is not dependent on insulin for glucose transport nor is there the enzymatic capacity for cholesterol synthesis, esterification a n d phospholipid ~ynthesis.~ I t has been suggested that the alterations seen in erythrocytes of diabetics may be, in part, due to disturbances in membrane fluidity.' Membrane fluidity is a characteristic of the lipid bilayer and is influenced by factors such as the relative concentrations of cholesterol and phospholipid, composition of phospholipid and
length and saturation ofcomponent fatty acids within the membrane.' Protein constituents of the membrane may also influence the fluidity of the lipid bilayer. This report forms part of a study of the relationships between the diabetic state and the physical properties of erythrocytes and presents results of membrane fluidity studies in a group of young diabetic subjects of varying metabolic control.
MATERIALS AND METHODS Subject5 Twenty subjects with type I diabetes mellitus aged 9.0 to 18.5 yr and with duration o f diabetes of 2 wk to 17 yr were studied. The group included children with evidence of early microvascular disease as demonstrated by retinal fluorescein angiogrdphy and 24-h urinary albumin excretion. Fluorescein angiography showed the presence of microaneurysms, hemorrhages and fluorescein leakage in varying degree in 6 of the 11 subjects studied by this method. Urinary albumin excretion ranged between 3 and 53 mg/l00 ml. The group showed wide variation in metabolic control as shown by fasting glucose levels (4.3-19.8 mmol,l), glycosylated hemoglobin levels (10.7-18.5:, HbA,). plasma triglyceride concentration (0.60-3.10 mmolil) and plasma cholesterol concentrations ( 3 . 4 7 . 2 mmol,l).
Methods Membrane fluidity was measured by fluorescence polarizatlon using the fluorescent probe 1.6-diphenyl-1.3.5-hexatriene(DPH). Hemoglobinfree ghosts were prepared by the method of Dodge et al.'" and resuspended at a concentration of approximately 50 pg proteinlml 5 mM phosphate buffer pH 7.4. Erythrocyte ghosts were labelled with an equal volume of 1 x lO-&MDPH and the resulting dispersion was left for 1 h at room temperature whilst the probe entered the hydrophobic portion of the membrane. Fluorescence polarization was measured in an Hitachi-Perkin-Elmer MPF3 spectrofluorometer equipped with a thermostated cell block and polarization accessory. Polarization data were calculated according to the formula - 11),(111+ I,)." Measurements were taken at 23OC, and P = (Ill paired normal samples were analyzed with test samples to minimlze batch variation. Control samples were obtained from healthy, nondiabetic laboratory staff. Erythrocyte membrane lipids were extracted by the method of Rose & Oaklander.L' The resultant extracts were assayed for cholesterol'? and pho~pholipidl~ content. Protein concentrations were estimated by the method of Lowry15 as modified by Peterson.Ih Glycosylated hemoglobin (HbA,) was determined by ion exchange chromatography on Bio-Rex 70 cation exchange resin. Blood glucose, plasma cholesterol and plasma triglyceride concentrations were measured by standard enzymatic methods. Urinary albumin excretion was measured by radioimmunoassay.17
450
Pathology (1983). 15. October
HILL & COURT
RESULTS N o difference in the degree of fluorescence polarization of D P H was found between diabetics and controls. In addition, no differences were detected in membrane phospholipid or cholesterol content and cholesterol phospholipid molar ratio of the diabetic subjects compared to controls. Results of fluorescence polarization of DPH and membrane cholesterol and phospholipid content are shown in Table 1. No correlations were found between age, sex, duration of diabetes, degree of metabolic control and presence o r absence of microvascular disease when with the level of fluorescence polarization.
ACKNOWLEDGEMENTS We are grateful to D r W. Sawyer, Biochemistry School, Melbourne University, for the use of the spectrofluorometer. This work was supported in part by grants from the Royal Children’s Hospital Research Foundation and the John Claude Kellion Foundation. Address f i j r correspondence: M.A.H., Department of Developmental Paediatrics, Royal Children’s Hospital, Parkville. Victoria, Australia 3052
Refe re nces
DISCUSSION It has been suggested that the abnormal rheology previously described in diabetic erythrocytes may be due to alterations in the membrane lipid b i l a ~ e r .Using ~ diphenyl hexatriene as a fluorescent probe of the hydrophobic portion of the membrane bilayer we have been unable to detect any differences in polarization between the diabetic group and controls. This would appear to be supported by the observation that cholesterol and phospholipid concentrations were the same in both groups; cholesterol phospholipid ratio being a major determinant of membrane fluidity.’ These data differ from the study of Baba et al.7 who showed elevated microviscosity (microviscosity being calculated from fluorescence polarization data by the Perrin equationfor details see reference I I ) , in a group of mostly type 11 diabetics with fasting blood glucose concentrations greater than 7.8 mmol;l. This elevation in microviscosity was found in the presence of normal membrane cholesterol phospholipid ratios. The reason for the difference in observations in our study is not apparent, but it may be due to the different study group (type 1 diabetes), age difference, o r different level of diabetic control. The present study suggests that changes in erythrocyte membrane fluidity d o not play a major part in the TABLEI
alterations of the physical properties of blood seen in type 1 diabetes mellitus.
1.Hill MA, Court JM, Mitchell G . Blood rheology and microalbuminuria in type I diabetes. Lancet 1982;2:985. 2. McMillan DE, Utterback NG. Puma JL. Reduced erythrocyte deformability in diabetes. Diabetes 1978;27:895-901. 3. Schmid-Schonbein H , Volger E. Red cell aggregation and red cell deformability in diabetes. Diabetes 1976;25(Supp1.2):897-902. 4.Wautier JL, Paton RC, Wautier MP et al. Increased adhesion of erythrocytes to endothelial cells i n diabetes mellitus and its relation to vascular complications. N Engl J Med 1980;305:237-41. 5. Chase HP, Glasgow AM. Juvenile diabetes mellitus and serum lipids and lipoprotein level. Am J Dis Child 1976;130:1I13-7. 6. Sosenko JM, Breslow JL. Miettinen 0s. Gabbay KH. Hyperglycemia and plasma lipid levels. N Engl J Med 1980;302:650-4. 7.Cooper RA. Decreased fluidity of red cell membrane lipids in abetalipoproteinemia. J Clin Invest 1977;60: 1 1 5 21. 8. Baba Y, Kai M, Kamda T et al. Higher levels of erythrocyte membrane microviscosity in diabetes. Diabetes 1979;28:1 138-40. 9. Cooper RA, Sawyer WH, Leslie MH ct al. Normal fluidity ofred cell membranes in hereditary spherocytosis Br J Haemol 1980;46:299-301. 10. Dodge JT, Mitchell C, Hanahan D. The preparation and chemical characteristics of haemoglobin free ghosts of human erythrocytes. Arch Biochem Biophys 1963; 100:119-30. 11. Shinitzky M, Barenholz Y . Fluidity parameters of lipid regions determined by fluorescence polarization. Biochem Biophys Acta 1978;515:367-94. 12. Rose HG. Oaklander M. Improvcd procedures for extraction of lipids from human erythrocytes. J Lipid Res 1965;6:428 - 31.
Fluorescence polarization and lipid data from erythrocyte membrane preparations
Fluorescence polarization Membrane cholesterol content (mg:ml packed cells) Membrane phospholipid content (mg:ml packed cells) Cholesterol!phospholipid (mol,mol)
Diabetics
Controls
Reference data for controls
0.240k0.006
0.243 k0.007
14.3k0.7
14.410.7
14.610.7’
31.4+2.2
30.9 t 1.5
3 1 . 2 i 1.6’
0.92 i 0 . 0 6
0.93 +0.08
0.95 k0.06’
Notes; Results are expressed as meanksd. Differences between control and diabetic values are not significant, as determined by Student t test.
MEMBRANE MICROVISCOSITY I N DIABETES
13. Zlatkis A, Bennie Z, Boyle AJ. A new method for the direct determination ofserum cholesterol. J Lab Clin Med 1953:41:486-92. 14. Bartlett G. Phosphorus assay in column chromatography. J Biol Chem 1950;234:486-92. 15. Lowry OH, Rosebrough N J , Farr AL, Randall RJ. Protein measurement with the fohn phenol reagent. J Biol Chem 1950: 193:265-75.
45 1
16. Peterson GL. Review of the fohn phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall. Anal Biochem 1979;I00:20 1-20. 17. Court J M , Dunlop ME. Urinary albumin excretion in diabetic children. Med Probl Paediatr 1975;12:307-11.
ERYTHROCYTE MEMBRANE FLUIDITY IN TYPE 1 DIABETES MELLITUS MICHAEL A. HILLAND J. M. COURT Department of Dewlopmental Paediatrics, Royal Children's Hospital, Melbourne
Summary Erythrocyte membrane fluidity was determined in a group of type 1 diabetics in varying metabolic control. No difference in membrane fluidity, as measured by fluorescence polarization of 1,6-diphenyI-l,3,5-hexatriene, was found between cells from diabeticsubjects. In addition, nodifference wasdetected in membrane phospholipid and cholesterol content or the ratio of cholesterol to phospholipid in ihe diabetic subjects when compared to controls. The present study suggests that changes in erythrocyte membrane fluidity do not play a major role in the alterations of the physical properties of blood seen in type 1 diabetes mellitus.
Key Mord.v. diabetes mellitus, microvascular disease. erythrocyte membrane fluidity
INTRODUCTION There is increasing speculation that alterations to flow characteristics within the microcirculation are involved in the development of the vascular complications ofdiabetes mellitus. Microvascular flow may be altered by changes in the physical properties of erythrocytes, and such cells from diabetic subjects have been shown to be less deformable' more prone to aggregation3 and more adhesive to endothelial surfaces4 compared to erythrocytes from a non-diabetic population. In type 1 diabetes there may be alterations in plasma concentrations of lipids related to the blood glucose levels achieved in diabetic control.5, The erythrocyte is susceptible to changes in concentration of glucose and lipids in plasma as it is not dependent on insulin for glucose transport nor is there the enzymatic capacity for cholesterol synthesis, esterification a n d phospholipid ~ynthesis.~ I t has been suggested that the alterations seen in erythrocytes of diabetics may be, in part, due to disturbances in membrane fluidity.' Membrane fluidity is a characteristic of the lipid bilayer and is influenced by factors such as the relative concentrations of cholesterol and phospholipid, composition of phospholipid and
length and saturation ofcomponent fatty acids within the membrane.' Protein constituents of the membrane may also influence the fluidity of the lipid bilayer. This report forms part of a study of the relationships between the diabetic state and the physical properties of erythrocytes and presents results of membrane fluidity studies in a group of young diabetic subjects of varying metabolic control.
MATERIALS AND METHODS Subject5 Twenty subjects with type I diabetes mellitus aged 9.0 to 18.5 yr and with duration o f diabetes of 2 wk to 17 yr were studied. The group included children with evidence of early microvascular disease as demonstrated by retinal fluorescein angiogrdphy and 24-h urinary albumin excretion. Fluorescein angiography showed the presence of microaneurysms, hemorrhages and fluorescein leakage in varying degree in 6 of the 11 subjects studied by this method. Urinary albumin excretion ranged between 3 and 53 mg/l00 ml. The group showed wide variation in metabolic control as shown by fasting glucose levels (4.3-19.8 mmol,l), glycosylated hemoglobin levels (10.7-18.5:, HbA,). plasma triglyceride concentration (0.60-3.10 mmolil) and plasma cholesterol concentrations ( 3 . 4 7 . 2 mmol,l).
Methods Membrane fluidity was measured by fluorescence polarizatlon using the fluorescent probe 1.6-diphenyl-1.3.5-hexatriene(DPH). Hemoglobinfree ghosts were prepared by the method of Dodge et al.'" and resuspended at a concentration of approximately 50 pg proteinlml 5 mM phosphate buffer pH 7.4. Erythrocyte ghosts were labelled with an equal volume of 1 x lO-&MDPH and the resulting dispersion was left for 1 h at room temperature whilst the probe entered the hydrophobic portion of the membrane. Fluorescence polarization was measured in an Hitachi-Perkin-Elmer MPF3 spectrofluorometer equipped with a thermostated cell block and polarization accessory. Polarization data were calculated according to the formula - 11),(111+ I,)." Measurements were taken at 23OC, and P = (Ill paired normal samples were analyzed with test samples to minimlze batch variation. Control samples were obtained from healthy, nondiabetic laboratory staff. Erythrocyte membrane lipids were extracted by the method of Rose & Oaklander.L' The resultant extracts were assayed for cholesterol'? and pho~pholipidl~ content. Protein concentrations were estimated by the method of Lowry15 as modified by Peterson.Ih Glycosylated hemoglobin (HbA,) was determined by ion exchange chromatography on Bio-Rex 70 cation exchange resin. Blood glucose, plasma cholesterol and plasma triglyceride concentrations were measured by standard enzymatic methods. Urinary albumin excretion was measured by radioimmunoassay.17
450
Pathology (1983). 15. October
HILL & COURT
RESULTS N o difference in the degree of fluorescence polarization of D P H was found between diabetics and controls. In addition, no differences were detected in membrane phospholipid or cholesterol content and cholesterol phospholipid molar ratio of the diabetic subjects compared to controls. Results of fluorescence polarization of DPH and membrane cholesterol and phospholipid content are shown in Table 1. No correlations were found between age, sex, duration of diabetes, degree of metabolic control and presence o r absence of microvascular disease when with the level of fluorescence polarization.
ACKNOWLEDGEMENTS We are grateful to D r W. Sawyer, Biochemistry School, Melbourne University, for the use of the spectrofluorometer. This work was supported in part by grants from the Royal Children’s Hospital Research Foundation and the John Claude Kellion Foundation. Address f i j r correspondence: M.A.H., Department of Developmental Paediatrics, Royal Children’s Hospital, Parkville. Victoria, Australia 3052
Refe re nces
DISCUSSION It has been suggested that the abnormal rheology previously described in diabetic erythrocytes may be due to alterations in the membrane lipid b i l a ~ e r .Using ~ diphenyl hexatriene as a fluorescent probe of the hydrophobic portion of the membrane bilayer we have been unable to detect any differences in polarization between the diabetic group and controls. This would appear to be supported by the observation that cholesterol and phospholipid concentrations were the same in both groups; cholesterol phospholipid ratio being a major determinant of membrane fluidity.’ These data differ from the study of Baba et al.7 who showed elevated microviscosity (microviscosity being calculated from fluorescence polarization data by the Perrin equationfor details see reference I I ) , in a group of mostly type 11 diabetics with fasting blood glucose concentrations greater than 7.8 mmol;l. This elevation in microviscosity was found in the presence of normal membrane cholesterol phospholipid ratios. The reason for the difference in observations in our study is not apparent, but it may be due to the different study group (type 1 diabetes), age difference, o r different level of diabetic control. The present study suggests that changes in erythrocyte membrane fluidity d o not play a major part in the TABLEI
alterations of the physical properties of blood seen in type 1 diabetes mellitus.
1.Hill MA, Court JM, Mitchell G . Blood rheology and microalbuminuria in type I diabetes. Lancet 1982;2:985. 2. McMillan DE, Utterback NG. Puma JL. Reduced erythrocyte deformability in diabetes. Diabetes 1978;27:895-901. 3. Schmid-Schonbein H , Volger E. Red cell aggregation and red cell deformability in diabetes. Diabetes 1976;25(Supp1.2):897-902. 4.Wautier JL, Paton RC, Wautier MP et al. Increased adhesion of erythrocytes to endothelial cells i n diabetes mellitus and its relation to vascular complications. N Engl J Med 1980;305:237-41. 5. Chase HP, Glasgow AM. Juvenile diabetes mellitus and serum lipids and lipoprotein level. Am J Dis Child 1976;130:1I13-7. 6. Sosenko JM, Breslow JL. Miettinen 0s. Gabbay KH. Hyperglycemia and plasma lipid levels. N Engl J Med 1980;302:650-4. 7.Cooper RA. Decreased fluidity of red cell membrane lipids in abetalipoproteinemia. J Clin Invest 1977;60: 1 1 5 21. 8. Baba Y, Kai M, Kamda T et al. Higher levels of erythrocyte membrane microviscosity in diabetes. Diabetes 1979;28:1 138-40. 9. Cooper RA, Sawyer WH, Leslie MH ct al. Normal fluidity ofred cell membranes in hereditary spherocytosis Br J Haemol 1980;46:299-301. 10. Dodge JT, Mitchell C, Hanahan D. The preparation and chemical characteristics of haemoglobin free ghosts of human erythrocytes. Arch Biochem Biophys 1963; 100:119-30. 11. Shinitzky M, Barenholz Y . Fluidity parameters of lipid regions determined by fluorescence polarization. Biochem Biophys Acta 1978;515:367-94. 12. Rose HG. Oaklander M. Improvcd procedures for extraction of lipids from human erythrocytes. J Lipid Res 1965;6:428 - 31.
Fluorescence polarization and lipid data from erythrocyte membrane preparations
Fluorescence polarization Membrane cholesterol content (mg:ml packed cells) Membrane phospholipid content (mg:ml packed cells) Cholesterol!phospholipid (mol,mol)
Diabetics
Controls
Reference data for controls
0.240k0.006
0.243 k0.007
14.3k0.7
14.410.7
14.610.7’
31.4+2.2
30.9 t 1.5
3 1 . 2 i 1.6’
0.92 i 0 . 0 6
0.93 +0.08
0.95 k0.06’
Notes; Results are expressed as meanksd. Differences between control and diabetic values are not significant, as determined by Student t test.
MEMBRANE MICROVISCOSITY I N DIABETES
13. Zlatkis A, Bennie Z, Boyle AJ. A new method for the direct determination ofserum cholesterol. J Lab Clin Med 1953:41:486-92. 14. Bartlett G. Phosphorus assay in column chromatography. J Biol Chem 1950;234:486-92. 15. Lowry OH, Rosebrough N J , Farr AL, Randall RJ. Protein measurement with the fohn phenol reagent. J Biol Chem 1950: 193:265-75.
45 1
16. Peterson GL. Review of the fohn phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall. Anal Biochem 1979;I00:20 1-20. 17. Court J M , Dunlop ME. Urinary albumin excretion in diabetic children. Med Probl Paediatr 1975;12:307-11.