Britirh]otrmal of Haernatology, 1976, 32, 235.
The Significance of Plasma Lipoproteins on Erythrocyte Aggregation and Sedimeiitation R. SCHERER, A. MORARESCU AND G. RUHENSTROTH-BAUER
Max-Planck-Institut fir Biochemia, Martinsried bei Miinchen, West Germany (Received 16 M a y 197s; acceptedfor publication 1sJu1y 197s) SUMMARY. Increased erythrocyte aggregation can be induced by high concentrations of human lipoproteins. The dependence of aggregate formation on lipoprotein concentration was recorded by determination of erythrocyte sedimentation rate (ESR), by electrical measurement of the erythrocyte aggregation index (EAI) and by scanning electron microscopy. The lipoprotein concentrations necessary to induce a significantly increased ESR in an otherwise normal human plasma are much too high to be encountered in physiologic or even severe pathologic states. Therefore hyperlipoproteinaemia by itself cannot explain a raised ESR. In cases where the ESR is raised due to the presence of increased amounts of other erythrocyte aggregating plasma proteins (agglomerins), hyperlipoproteinaemia can contribute to a limited extent to the increase in ESR. The possible pathophysiological significance of the demonstrated erythrocyte aggregating capacity of human liproteins in a microvascular environment is noted. The influence of plasma lipids on erythrocyte sedimentation rate (ESR) has been a subject of clinical investigation almost since the introduction of the ESR determination as a routinc test into medical practice. While there are well-established inhibitory effects of lysolecithin and free fatty acids on ESR (Klibansky & De Vries, 1963 ; Schiebel & Ruhenstroth-Bauer, 1967; Adlkofer et a2, 1969; Berlin et al, 1973) reports on the significance of other plasma lipids on the ESR have been contradictory. Theorell (1930) claimed inhibitory effects of lecithin and cholesterol on the ESR, while Ohlson & Rundqvist (1932) reported that removal of plasma lipids by alcohol-ether extraction did not significantly alter the sedimentation rate in normal plasma or in plasma samples with increased sedimentation rate. In 1973 Bottiger et al found that the ESR was significantly raised in patients with asymptomatic hyperlipidaemia, although these authors pointed out that evidence for a direct causal relationship was missing. Most previous investigations on the influence of plasma lipids on the ESR were carried out with einulsions of the respective lipid class in plasma. This is no imitation of physiological or pathophysiological events, as plasma lipids even in severe hyperlipidaemia never occur as pure lipid emulsions, but always complexed to apoproteins forming liprotein classes of defined size and density with distinct physical and chemical properties. W e therefore decided Correspondence: Professor G. Ruhenstroth-Bauer, Max-Planck-Institut fiir Biochemie, D-So32 Martinsried bei Miinchen, West Germany.
23 s
23 6
R. Scherer, A. Morarescu and G. Ruhenstroth-Bauer
to study the effect of different concentrations of naturally occurring lipoproteins isolated from healthy human blood donors on erythrocyte aggregation and sedimentation. MATERIALS AND METHODS Fresh blood from healthy, fasting, young male blood donors was obtained from a local blood bank. After clot-formation and centrifugation potassium bromide was added to the serum to give a final density of 1.063 for the preparation of low-density and very-lowdensity lipoproteins and a density of 1.21 for the preparation of total lipoproteins (with the exclusion of very-high-density lipoproteins). After ultracentrifugation (Spinco, model L50 ultracentrifuge, Beckmann; 50 000 rpm, 104000 g for 24 h) the floating lipoproteins were removed and exhaustively dialysed against a buffer containing NaCl 0.9%, EDTA o.I%, pH 7.4. For the determination of the ESR different amounts of the dialysed lipoproteins were added to I ml of a solution of 1% human albumin (Behringwerke, Marburg, W. Germany) in NaCI/EDTA buffer. All solutions were concentrated to a volume of I ml with the aid of disposable Minicon B-concentrators (Amicon, Oosterhout) and washed group 0 erythrocytes were added to give a standardized haematocrit of 29.5%. The ESR was measured using Westergren tubes. The anhydrous weight of the lipoproteins was determined after extensive drying at 80°C. In another series of experiments human plasma from healthy, male blood donors was used instead of the human albumin solution. Before testing, all lipoproteins were removed by ultracentrifugation at density 1.21 and the plasmas were dialysed against NaClI EDTA buffer. Erythrocyte aggregation was measured electrically (Boss et al, 1975). The determination of the erythrocyte aggregation index (EAI) as measure of the aggregation is based on a technical modification of the Coulter apparatus as developed in our department (Kachel, 1972). For the measurement, the number of erythrocytes in the cell suspensions was reduced by I :300 as compared to the ESR tests, The red cell suspensions were submitted to a defined shear stress by rotating them for 5 min at 500 rpm, 750 rpm and 250 rpm respectively. After further Ioo-fold dilution with 2.5% dextran T 250 (Pharmacia, Uppsala, Sweden) in aqueous saline solution the percentage of single and aggregated cells was determined by volume classification after passage through a modified Coulter capillary (Kachel, 1972). The EAI was calculated from the three aggregation curves measured at different shear stress. Electrical measurement of erythrocyte aggregation by liproteins was performed with plasma containing increasing amounts of lipoproteins ( D< I .063). In a number of patients, where, together with elevated ESR, hyperlipoproteinaemia was diagnosed, the effect of removal of total lipoproteins on the ESR was studied. EDTA-plasma from these patients was diluted 1:12 with 3.8% sodium citrate, brought to density 1.21 by addition of potassium bromide and ultracentrifuged as described above. The control samples from the same patients were treated the same way, but at the end of the ultracentrifugation these tubes were shaken instead of removing the floating lipoproteins. After dialysis and concentration back to the original volume using a Minicon B concentrator, the ESR of the plasma samples (including the controls) was measured as described above. Reading of the ESR was performed when the control had sedimented past 50 mm. Measurements were
Erythrocyte Sedimentation
237
usually performed in duplicate on each patient. P-Lipoprotein concentration in the plasma samples of the patients was determined using Partigen immunodiffusionplates (Behringwerke, Marburg). The procedure of sampIe preparation for scanning electron microscopy was as follows: one drop of the same red cell suspension that was used for the ESR determination was put on a coverslip. This was subsequently rinsed very gently with a solution of 1% glutaraldehyde in isotonic phosphate-buffered saline, leaving enough red cells for microscopic examination attached to the coverslip. Fixation was continued in 1% glutaraldehyde for 30 min, followed by additional fixation in 0.5% osmium tetroxide. After washing the specimen briefly in distilled water dehydration was performed by subsequent washes in 50, 70, 80, go, 95 and 100% alcohol and finally in propylenoxide. This procedure with the red cells remaining attached to the coverslip was adopted in order to omit centrifugation which would have caused disruption of the red cell aggregates. After coating with gold, the specimens were examined with a Cambridge Mark IIa scanning electron microscope at 27 kV beam voltage.
Lipoproteins D
The Significance of Plasma Lipoproteins on Erythrocyte Aggregation and Sedimeiitation R. SCHERER, A. MORARESCU AND G. RUHENSTROTH-BAUER
Max-Planck-Institut fir Biochemia, Martinsried bei Miinchen, West Germany (Received 16 M a y 197s; acceptedfor publication 1sJu1y 197s) SUMMARY. Increased erythrocyte aggregation can be induced by high concentrations of human lipoproteins. The dependence of aggregate formation on lipoprotein concentration was recorded by determination of erythrocyte sedimentation rate (ESR), by electrical measurement of the erythrocyte aggregation index (EAI) and by scanning electron microscopy. The lipoprotein concentrations necessary to induce a significantly increased ESR in an otherwise normal human plasma are much too high to be encountered in physiologic or even severe pathologic states. Therefore hyperlipoproteinaemia by itself cannot explain a raised ESR. In cases where the ESR is raised due to the presence of increased amounts of other erythrocyte aggregating plasma proteins (agglomerins), hyperlipoproteinaemia can contribute to a limited extent to the increase in ESR. The possible pathophysiological significance of the demonstrated erythrocyte aggregating capacity of human liproteins in a microvascular environment is noted. The influence of plasma lipids on erythrocyte sedimentation rate (ESR) has been a subject of clinical investigation almost since the introduction of the ESR determination as a routinc test into medical practice. While there are well-established inhibitory effects of lysolecithin and free fatty acids on ESR (Klibansky & De Vries, 1963 ; Schiebel & Ruhenstroth-Bauer, 1967; Adlkofer et a2, 1969; Berlin et al, 1973) reports on the significance of other plasma lipids on the ESR have been contradictory. Theorell (1930) claimed inhibitory effects of lecithin and cholesterol on the ESR, while Ohlson & Rundqvist (1932) reported that removal of plasma lipids by alcohol-ether extraction did not significantly alter the sedimentation rate in normal plasma or in plasma samples with increased sedimentation rate. In 1973 Bottiger et al found that the ESR was significantly raised in patients with asymptomatic hyperlipidaemia, although these authors pointed out that evidence for a direct causal relationship was missing. Most previous investigations on the influence of plasma lipids on the ESR were carried out with einulsions of the respective lipid class in plasma. This is no imitation of physiological or pathophysiological events, as plasma lipids even in severe hyperlipidaemia never occur as pure lipid emulsions, but always complexed to apoproteins forming liprotein classes of defined size and density with distinct physical and chemical properties. W e therefore decided Correspondence: Professor G. Ruhenstroth-Bauer, Max-Planck-Institut fiir Biochemie, D-So32 Martinsried bei Miinchen, West Germany.
23 s
23 6
R. Scherer, A. Morarescu and G. Ruhenstroth-Bauer
to study the effect of different concentrations of naturally occurring lipoproteins isolated from healthy human blood donors on erythrocyte aggregation and sedimentation. MATERIALS AND METHODS Fresh blood from healthy, fasting, young male blood donors was obtained from a local blood bank. After clot-formation and centrifugation potassium bromide was added to the serum to give a final density of 1.063 for the preparation of low-density and very-lowdensity lipoproteins and a density of 1.21 for the preparation of total lipoproteins (with the exclusion of very-high-density lipoproteins). After ultracentrifugation (Spinco, model L50 ultracentrifuge, Beckmann; 50 000 rpm, 104000 g for 24 h) the floating lipoproteins were removed and exhaustively dialysed against a buffer containing NaCl 0.9%, EDTA o.I%, pH 7.4. For the determination of the ESR different amounts of the dialysed lipoproteins were added to I ml of a solution of 1% human albumin (Behringwerke, Marburg, W. Germany) in NaCI/EDTA buffer. All solutions were concentrated to a volume of I ml with the aid of disposable Minicon B-concentrators (Amicon, Oosterhout) and washed group 0 erythrocytes were added to give a standardized haematocrit of 29.5%. The ESR was measured using Westergren tubes. The anhydrous weight of the lipoproteins was determined after extensive drying at 80°C. In another series of experiments human plasma from healthy, male blood donors was used instead of the human albumin solution. Before testing, all lipoproteins were removed by ultracentrifugation at density 1.21 and the plasmas were dialysed against NaClI EDTA buffer. Erythrocyte aggregation was measured electrically (Boss et al, 1975). The determination of the erythrocyte aggregation index (EAI) as measure of the aggregation is based on a technical modification of the Coulter apparatus as developed in our department (Kachel, 1972). For the measurement, the number of erythrocytes in the cell suspensions was reduced by I :300 as compared to the ESR tests, The red cell suspensions were submitted to a defined shear stress by rotating them for 5 min at 500 rpm, 750 rpm and 250 rpm respectively. After further Ioo-fold dilution with 2.5% dextran T 250 (Pharmacia, Uppsala, Sweden) in aqueous saline solution the percentage of single and aggregated cells was determined by volume classification after passage through a modified Coulter capillary (Kachel, 1972). The EAI was calculated from the three aggregation curves measured at different shear stress. Electrical measurement of erythrocyte aggregation by liproteins was performed with plasma containing increasing amounts of lipoproteins ( D< I .063). In a number of patients, where, together with elevated ESR, hyperlipoproteinaemia was diagnosed, the effect of removal of total lipoproteins on the ESR was studied. EDTA-plasma from these patients was diluted 1:12 with 3.8% sodium citrate, brought to density 1.21 by addition of potassium bromide and ultracentrifuged as described above. The control samples from the same patients were treated the same way, but at the end of the ultracentrifugation these tubes were shaken instead of removing the floating lipoproteins. After dialysis and concentration back to the original volume using a Minicon B concentrator, the ESR of the plasma samples (including the controls) was measured as described above. Reading of the ESR was performed when the control had sedimented past 50 mm. Measurements were
Erythrocyte Sedimentation
237
usually performed in duplicate on each patient. P-Lipoprotein concentration in the plasma samples of the patients was determined using Partigen immunodiffusionplates (Behringwerke, Marburg). The procedure of sampIe preparation for scanning electron microscopy was as follows: one drop of the same red cell suspension that was used for the ESR determination was put on a coverslip. This was subsequently rinsed very gently with a solution of 1% glutaraldehyde in isotonic phosphate-buffered saline, leaving enough red cells for microscopic examination attached to the coverslip. Fixation was continued in 1% glutaraldehyde for 30 min, followed by additional fixation in 0.5% osmium tetroxide. After washing the specimen briefly in distilled water dehydration was performed by subsequent washes in 50, 70, 80, go, 95 and 100% alcohol and finally in propylenoxide. This procedure with the red cells remaining attached to the coverslip was adopted in order to omit centrifugation which would have caused disruption of the red cell aggregates. After coating with gold, the specimens were examined with a Cambridge Mark IIa scanning electron microscope at 27 kV beam voltage.
Lipoproteins D
